The Physiology of Multifactorial Problems Limiting the ... · Workshop 1:Fisiologia reprodutiva bovina: conhecimentos aplicados para otimizar a fertilidade e o melhoramento genético

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Acta Scientiae Veterinariae. 38(Supl 2): s277-s315, 2010.

ISSN 1678-0345 (Print)ISSN 1679-9216 (Online)

The Physiology of Multifactorial Problems Limiting the Establishmentof Pregnancy in Dairy Cows

A.C.O. Evans1, E.J. Williams1 & S.W. Walsh1

ABSTRACT

Background: Dairy production systems that have benefited from huge increases in milk production in recent decadeshave suffered a decline in fertility. The causes, and consequently the solutions, to this are both genetic and managementin origin.

Review: We consider the following points have the greatest negative impact on fertility and need to be addressed inany effort to ameliorate the problem. Firstly, timely resolution of any infection of the post partum uterus followed byexpression and detection of oestrus and insemination with high quality semen (Day 0). This must be followed by theovulation and fertilization of a high quality oocyte (Day 1). After ovulation an early increase in progesterone secretionfrom the corpus luteum (Days 3 to 7) is needed. This and other factors are then needed to ensure that the uterineendometrium produces an early and appropriate environment to stimulate embryo development (Day 6 to 13). Thisleads to the growth of a large embryo producing adequate quantities of interferon tau (Day 14 to 18) to alter uterineprostaglandin secretion and signal maternal recognition of pregnancy (Day 16 to 18).

Conclusion: Improvements to any of these events will achieve important increases in fertility but in combination thepossibility exists to greatly benefit reproduction and the efficiency of dairy production systems.

Keywords: cows, dairy production, fertility.

1School of Agriculture Food Science and Veterinary Medicine, University College Dublin, Ireland. CORRESPONDENCE: A.C.O. Evans[[email protected].]Supported by Science Foundation Ireland (07/SRC/B1156).

www.ufrgs.br/favet/revista

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Workshop 1:Fisiologia reprodutiva bovina: conhecimentos aplicados para otimizar a fertilidade e o melhoramentogenético. Acta Scientiae Veterinariae. 38 (Supl 2): s277-s315

Simplification of superovulation protocols in cattle*

Gabriel A. Bó1,2,5, Andrés Tríbulo1,2, Martín Ramos1,2, Daniel Carballo Guerrero1,2, RicardoTribulo1,2, Humberto Tribulo1,2, Dragan Rogan3 & Reuben J. Mapletoft4

ABSTRACT

Background: Successful bovine embryo transfer programs require the use of simple superovulation protocols andhigh numbers of transferable embryos. The control of follicular wave emergence and ovulation have facilitated donormanagement, but the most commonly used treatment, estradiol, cannot be used in many parts of the world, andmechanical removal of the dominant follicle is difficult to apply in the field. Other alternatives include GnRH or LH, butefficacy in groups of randomly cycling animals is variable.

Review: An alternative treatment to control follicular wave emergence is to increase the response to GnRH byinducing a persistent follicle and initiating FSH treatments following GnRH-induced ovulation. The number of transferableembryos following superovulation during the first follicular wave arising at the time of the GnRH-induced ovulation didnot differ from that achieved 4 days after treatment with estradiol benzoate and progesterone. To further simplifysuperovulation, FSH has been diluted in a slow-release formulation (SRF) and administered as a single or a splitintramuscular injection. Although, a single intramuscular injection of Folltropin-V in SRF was highly efficacious in theinduction of superovulation in a variety of breeds of beef cattle, it was difficult to mix with Folltropin-V. However, in asubsequent series of experiments it was shown that reducing the initial concentration of SRF to 25% and administeringthe Folltropin-V as two intramuscular injections 48 hours apart (called split-single administration) facilitated the dilutionof Folltropin-V with the SRF and resulted in a superovulatory response that did not differ from controls.

Conclusion: The incorporation of GnRH-based protocol to control follicular dynamics and ovulation have the advantageof being able to schedule the treatments quickly and without the need for detecting estrus in donor cows. The single-split intramuscular injection of Folltropin-V in 25% SRF has the potential to reduce labor and handling and may beuseful when handling stress is an impediment to success. These treatments are practical and easy to perform by thefarm staff, facilitating the widespread application of embryo transfer technologies.

Keywords: bovine, superovulation, estradiol, GnRH, FSH.

*Research was supported by the Instituto de Reproducción Animal Córdoba (IRAC) and Bioniche Animal Health (Canada). We alsothank Biotay SA (Argentina) for the provision on PGF2á and GnRH and facilitating the importation of the other hormones used in thesetrials. Special thanks to our colleagues of IRAC for technical assistance. 1Instituto de Reproducción Animal Córdoba (IRAC), Zona RuralGeneral Paz, (5145) Córdoba, Argentina. 2Facultad de Ciencias Agropecuarias, Universidad Nacional de Córdoba, Argentina. 3BionicheLife Sciences, Belleville, ON, Canada. 4Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada.5Corresponding author, CORRESPONDENCE: [E-mail: [email protected]]

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I. INTRODUCTION

II. MANIPULATION OF THE FOLLICULAR WAVE FOR SUPEROVULATION

III. SUPEROVULATION DURING THE FIRST FOLLICULAR WAVE SUPEROVULATIONDURING THE FIRST FOLLICULAR WAVE AFTER GNRH

IV. SUPEROVULATION WITH ONE OR TWO FSH INJECTIONS SUPEROVULATION WITHA SINGLE ADMINISTRATION OF FSH SUPEROVULATION WITH A SPLITADMINISTRATION OF FSH

V. CONCLUSIONS

I. INTRODUCTION

Variability in the response to superovulation protocols and the time and effort required to administer treatmentshave affected the widespread application of embryo transfer in genetic improvement programs [10]. Although researchefforts in recent years have resulted in no increase in the number of transferable embryos per treatment, protocolsthat control emergence of the follicular wave [7,8] and the timing of ovulation [2,9] have allowed the treatment ofgroups of donors, regardless of the stage of the estrous cycle and permitted fixed-time artificial insemination (FTAI)of donors, without the need to detect estrus. This has had a positive impact on commercial embryo transfer, becauseit has facilitated the scheduling of working protocols, without being dependent on the knowledge and skill of personnelto detect estrus. However, the complexity of protocols can lead to errors that can affect superovulatory response andembryo production. In this regard, the need to inject FSH twice daily is a factor of great concern [6]. Moreover, themost commonly used treatment for synchronization of follicular wave emergence for superovulation involves the useof a progestogen/progesterone (P4) releasing device and estradiol-17β or its esters (E

2) [33], which cannot be used in

many countries because of concerns about the effects of steroid hormones in the food chain [27]. This review is anupdate to one published previously [11]. It presents progress in the development of superovulation protocols usingGnRH as a replacement for E

2 to synchronize follicular development, and the administration of FSH in one or two

injections as an alternative to the traditional superovulation treatment of eight FSH injections given twice daily.

II. MANIPULATION OF THE FOLLICULAR WAVE FOR SUPEROVULATION

Traditionally, gonadotrophin treatments were initiated during the mid-luteal phase, approximately 9 to 11days after estrus [29], around the time of emergence of the second follicular wave [24]. However, a greatersuperovulatory response occurred when treatments were initiated on the day of follicular wave emergence, ratherthan 1 day before, or 1 or 2 days later [1,38]. Therefore, conventional treatment protocols have two drawbacks: 1) therequirement to have trained personnel dedicated to the detection of estrus, and 2) the necessity to have all donors inestrus at the same time in order to initiate treatments at the same time.

In the 1990’s, we reported on the use of P4 and E2 to synchronize follicular wave emergence, on average,

4 days after treatment [7,9]. This treatment has been used by practitioners around the world [2,9,33], but its use hasbeen restricted recently in several countries. This restriction leaves many embryo transfer practitioners with a seriousdilemma and created the need to develop treatments that do not involve the use of E

2.

An alternative is to eliminate the suppressive effect of the dominant follicle by ultrasound-guided follicleaspiration and initiate superstimulatory treatments 1 or 2 days later [5,12]. The disadvantage is that this procedurerequires ultrasound equipment and trained personnel which is only appropriate for embryo production centers, wheredonors are held; it is very difficult to apply in the field.

Another alternative to synchronize follicular wave emergence is to use LH or GnRH to induce ovulation ofthe dominant follicle [32] which is followed by wave emergence 1 to 2 days later [40]. However, this occurs by chance

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(stage of the cycle) or only when the treatment results in ovulation [34]. Pursley et al. [40] reported that the administrationof GnRH at random stages of the cycle induced ovulation in about 85% of dairy cows, but more recently, Colazo et al.[19] reported an ovulation rate of 62.4% in lactating dairy cows receiving 25 mg pLH (Lutropin-V, Bioniche AnimalHealth, Belleville, ON, Canada) and 44.3% in those receiving 100 µg GnRH. In another study, pLH induced ovulationin 78% and GnRH induced ovulation in 56% of beef heifers [34]. The occurrence of ovulation following administrationof GnRH or pLH in beef cows appears to be around 60% [43]. Not surprisingly, treatment with GnRH at random stagesof the estrous cycle prior to initiating superstimulatory treatments resulted in lower responses than treatments initiatedafter follicular aspiration or E

2 treatment [21]. However, in a retrospective analysis of commercial data, Hinshaw

[personal communication; AETA 2007] found no differences in the number of transferable embryos between donorssuperstimulated 4 days after treatment with E

2 and P4 (7.8 transferable embryos, n = 1136) and those superstimulated

2 days after treatment with GnRH (7.7 transferable embryos, n = 56). In another recent study [49], CIDR-treated dairycows (n = 411) were superstimulated 4 days after receiving E

2 or 2 days after GnRH to synchronize follicular wave

emergence; there was no significant difference in the number of ova/embryos or transferable embryos betweengroups. In another retrospective analysis of commercial data, dairy donors superstimulated 60 hours after theadministration of GnRH (n = 245) produced similar numbers of transferable embryos as those superstimulated 4 daysafter receiving E

2 (n = 691) [44]. Controlled studies with the use of GnRH must be conducted to validate these

promising results.

III. SUPEROVULATION DURING THE FIRST FOLLICULAR WAVE

Follicular wave emergence occurs consistently at the time of ovulation [24], and experiments performed incattle [38] and sheep [35] have indicated that it is possible induce superovulation of follicles in the first follicular wave.Adams et al. [1] also reported no difference in superovulatory response when FSH treatments were initiated at thetime of emergence of the first or second follicular wave. However, success relies upon successful determination ofthe time of ovulation or accurate estrus detection, with ovulation expected to occur 1 day after the onset of estrus.

To avoid the need to observe estrus and ovulation in Nelore (Bos indicus) donors, Nasser et al. [39] inducedsynchronous ovulation with a protocol that involved the administration of E

2 at the time of P4 (CIDR) insertion (Day 0),

prostaglandin F2α (PGF

2α) at CIDR removal (Day 8) and followed by pLH 24 hours later. FSH treatments were initiated24 hours after pLH (i.e., the expected time of ovulation and emergence of the first follicular wave). Superovulatoryresponse did not differ from a contemporary group superstimulated 4 days after treatment with E

2. However, the

number of transferable embryos was reduced in cows superstimulated during the first follicular wave without anaccompanying use of a CIDR.

Superovulation during the first follicular wave after GnRH

Recently, we conducted a series of five experiments with the overall objective of developing a protocol forsuperovulation during the first follicular wave, using P4-releasing devices but not E

2. We considered previous reports

indicating that ovulatory response to GnRH could be increased by the administration of PGF2á

to regress the CL at thetime of insertion of a P4-releasing device that remained in place for 7 to 10 days [43]; ovulation and wave emergenceoccurred 1 to 2 days after the administration of GnRH [43].

In the first experiment [14], 70 Bonsmara donors (29 cows and 41 heifers) were randomly assigned to oneof two treatment groups. Donors in the First Wave group received a P4-releasing device (1.56 g of P4, Cue-Mate,Bioniche Animal Health) along with a dose of PGF2a (0.150 mg D (+) cloprostenol, Bioprost-D, Biotay SA, Argentina)at random stages of the estrous cycle. Cue-Mates were removed 10 days later and a second PGF

2a was administered,

followed by GnRH (0.050 mg Lecirelina, Biosin-OV, Biotay SA) 36 hours later. Ovulation was expected to occur within36 hours after GnRH. On Day 0 (36 hours after GnRH), donors received a new Cue-Mate and treatment with a totaldose of 200 to 260 mg (heifers) or 320 mg (cows) NIH-FSH-P1 of Folltropin-V (Bioniche Animal Health) in twice dailydecreasing doses over 5 days was initiated. Donors received PGF

2a with the last two Folltropin-V treatments and Cue-

Mates were removed with the last treatment. All donors received 12.5 mg Lutropin-V 24 hours after Cue-Mate removaland were FTAI 12 and 24 hours later. Ova/embryos were collected 7 days later. Donors in the Control group receiveda Cue-Mate and E

2 (Bioestradiol, Biotay SA) and 50 mg of P4 (Progesterona Río de Janeiro, Laboratorios Allignani

Hnos SRL, Argentina) and Folltropin-V treatments were initiated 4 days later with similar dosages and treatment

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protocol as the First Wave group. The mean (±SEM) number of transferable embryos did not differ significantlybetween the Control (5.1 ± 0.9) and First Wave (3.7 ± 0.8) groups.

Although this study indicated that superovulation during the first follicular wave was as efficacious as the“standard” E2+P4 treatment protocol in beef cattle, the duration of the protocol (26 days vs 15 days from Cue-Mateinsertion until embryo collection) made it time-consuming and difficult to implement. Therefore, more studies weredesigned to shorten and simplify the protocol. In the second study [15], superovulatory response was comparedbetween donors that were pretreated with a Cue-Mate for 10 days vs those pretreated with a Cue-Mate for 5 days.Ovulation rate and the interval from GnRH administration to ovulation was 81.8% (9/11) and 32.0 ± 2.8 hours vs 100%(11/11) and 36.0 ± 0.0 hours, respectively. The number to transferable embryos did not differ between groups (5.5 ±1.6 vs. 5.2 ± 2.6 for cows pretreated with a Cue-Mate for 10 vs 5 days, respectively) and neither differed from donorssuperstimulated 4 days after E

2 + P4 (5.4 ± 1.6).

A third experiment [16] determined whether it was necessary to remove the P4-releasing device during thesuperovulation protocol. Angus cows (n = 27) and heifers (n = 10) were superovulated by two different treatmentprotocols in a cross over design. All donors received a Cue-Mate along with PGF

2á, at random stages of the estrous

cycle; those in Group 1 received a second PGF2á

at the time of Cue-Mate removal 5 days later, followed by GnRH 36hours later. On Day 0 (36 hours after GnRH) donors received a new Cue-Mate and superovulation treatments wereinitiated with a total dose of 400 mg NIH-FSH-P1 of Folltropin-V in twice daily decreasing doses over 5 days. PGF

2á

administration, Cue-Mate removal, pLH administration, FTAI and embryo collections were done as in previous studies.Donors in Group 2 were treated similarly to those in Group 1, except that Cue-Mates were not removed but remainedin place for 13 days (i.e., were removed with the last FSH and PGF

2á injections). Ovulation rate and the interval from

GnRH treatment to ovulation was 86.5% (64/74) and 35.6±1.6 hours for donors in which the Cue-Mate was replacedduring the treatment (Group 1) and 89.2% (33/37) and 37.5 ± 0.7 hours for those in which the Cue-Mate was notreplaced (Group 2). Mean (± SEM) numbers of ova/embryos and transferable embryos were 8.2 ± 1.0 and 4.1 ± 0.6vs 9.8 ± 0.9 and 5.7 ± 0.7 for Groups 1 and 2, respectively (P>0.2). It was not necessary to remove the P4-releasingdevice to synchronize ovulation (and follicle wave emergence for superovulation) with GnRH.

A fourth experiment [16] evaluated the efficacy of shortening the protocol one more day, by giving Folltropin-V for 4 rather than 5 days, as in the previous three experiments. Simmental (n = 18) and Angus (n = 6) cows weresuperstimulated by two different treatment protocols in a cross over design. Cows in both groups were treatedsimilarly to those in Group 2 in the previous experiment (i.e., Cue-Mates were not replaced during treatment). Cows inGroup 1 (control) received FSH over 5 days; while those in Group 2 received the same dosage of Folltropin-V, butgiven in twice daily decreasing doses over 4 days (Cue-Mates were removed with the last FSH and PGF

2á injections).

Mean (± SEM) numbers of ova/embryos and transferable embryos were 13.5 ± 2.4 and 6.6 ± 1.1 vs 12.0 ± 1.9 and5.8 ± 1.0 for Groups 1 and 2, respectively (P>0.6).

A final experiment determined whether it was possible to reduce the number of PGF2á

treatments during thepre-treatment, further simplifying the first wave protocol [17]. A second objective was to confirm the effectiveness ofthe first wave protocol by comparing it with the P4 + E

2 protocol to synchronize follicle wave emergence. Simmental

donors cows (n = 14) were assigned randomly to three treatment groups in a cross-over design so that all animalsreceived all treatments. Donors in Groups 1 and 2 received Cue-Mates as in the previous experiments, but those inGroup 1 received PGF

2á at the time of Cue-Mate insertion and 5 days later (as in previous experiments), whereas

those in Group 2 received PGF2á

at Cue-Mate insertion only (i.e., eliminating the need to handle animals on Day 5).Cows in Groups 1 and 2 received GnRH 7 days after Cue-Mate insertion to induce ovulation and treatments withFolltropin-V were initiated 36 hours later (Day 0; as in previous experiments). Donors in Group 3 received a Cue-Mateplus P4 + E

2 at random stages of the estrous cycle and FSH treatments were initiated 4 days later. All cows received

400 mg of Folltropin-V in twice daily injections over 4 days as described in the previous experiment. Mean (± SEM)numbers of ova/embryos and transferable embryos did not differ among groups (12.9 ± 2.0 and 6.6 ± 1.2, 11.5 ± 1.7and 7.7 ± 1.6, and 14.5 ± 2.8 and 6.8 ± 1.7 for Groups 1, 2 and 3, respectively).

In conclusion, the results from these studies indicate that superovulation during the first follicular waveafter inducing ovulation with GnRH can be used successfully in groups of randomly cycling donors without the needfor estrus detection or E

2 to synchronize follicular wave emergence. The protocol is easy to follow and embryo

production is comparable to that of the E2 and P4 protocol. The recommended protocol consists of the administration

of PGF2á

at the time of Cue-Mate insertion (Day 0), followed by the administration of GnRH on Day 7 AM. Superovulation

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treatments are then initiated on Day 8 PM (36 hours after GnRH), with twice-daily injections of FSH until Day 12 AM,PGF

2á is administered on Days 11 PM and 12 AM and Cue-Mates are removed on Day 12 AM; GnRH or pLH is given

on Day 13 AM and donors are FTAI 12 and 24 hours later. Finally, embryos are collected on Day 20. If a practitionerprefers to use a 5-day instead of a 4-day FSH treatment protocol, the last FSH and PGF

2á injections and Cue-Mate

removal are done on Day 13 AM (instead of Day 12 AM), pLH or GnRH is given on Day 14 AM with FTAI 12 and 24hours later and embryos are collected on Day 21. This protocol can be easily organized on a calendar as is shown inFigure 1.

IV. REDUCING THE NUMBER OF FSH ADMINISTRATIONS IN A SUPEROVULATIONPROTOCOL

Superovulation with a single administration of FSH

Traditional superstimulatory treatments consist of a single administration of equine chorionic gonadotrophin(eCG) or twice daily injections of pituitary extracts containing FSH over 4 or 5 days [33]. The eCG is a complexglycoprotein which has a long half-life (over 40 hours) which represents a practical advantage, since a singleadministration will induce ovarian superovulation [37,42]. However, it is necessary to neutralize eCG with antibodiesat the time of insemination to avoid the adverse effects of continuing ovarian stimulation (large unovulated follicles atthe time of embryo collection and decreased embryo collection efficiency and quality); [reviewed in 22]. In contrast,the half-life of FSH is 5 hours in the cow [20,28] and requires frequent applications to induce superovulation [4,36].Twice daily treatments with FSH have resulted in a greater superovulatory response than once daily administration[30,36,48].

The need to inject FSH twice a day requires frequent attention by farm-personnel and increases the possibilityof failures due to mishandling and errors in administration of treatments. In addition, twice daily treatments may causeundue stress in donor cows with a subsequent decreased superovulatory response [6,23], and/or altered preovulatory

Figure 1. Treatment schedule for superovulation of donor cows during the first follicular wave. Donors receive a progesterone-releasingdevice (Cue-Mate) along with PGF2á followed by GnRH 7 days later. On Day 0 [36 hours after GnRH or Lutropin-V (pLH)], superovulationwith Folltropin-V (FSH) is initiated (twice daily decreasing doses over 4 days). PGF2á is administered with the last two FSH injections andCue-Mate are removed with the last FSH injection. Ovulation is induced with GnRH or pLH 24 hours after Cue-Mate removal, donors arefixed-time inseminated (AI) 12 and 24 hours later and ova/embryos are collected 7 daysafter pLH treatment [Adapted from Carballo et al.2010, 17].

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LH surge [45]. Therefore, simplified protocols of superovulation may be expected to reduce donor handling costs andimprove response, particularly in less tractable animals.

More than 10 years ago, we reported that a single subcutaneous administration of 400 mg NIH-FSH-P1 ofFolltropin-V in beef cows with good body condition (>3 out of 5), resulted in a superovulatory response equivalent tothe traditional treatment protocol of twice daily injections over 4 days [6]. However, the results could not be repeatedin Holstein cows, which had less subcutaneous fat [25]. In a subsequent study in Holstein cows, the single injectionwas split into two, with 75% of the dose of Folltropin-V administered subcutaneously on the first day of treatment andthe remaining 25% administered 48 hours later, when PGF

2α is normally administered [31]. Superovulatory responsewas intermediate, between that with the traditional protocol (the highest response) and a single subcutaneous injection(the lowest response).

An alternative to induce a consistent superovulatory response with a single injection of FSH would be tocombine the pituitary extract with agents that cause the hormone to be released slowly over several days. Theseagents are commonly known as polymers, are biodegradable and non-reactive in the tissue, facilitating use in animals[46]. Yamamoto et al. [50] reported that FSH in a 30% solution of polyvinylpyrrolidone (PVP) and administered in asingle intramuscular injection resulted in a comparable superovulatory response to twice daily treatments. However,Callejas et al. [13] and Bó & Mapletoft (unpublished observations) were unable to induce a satisfactory superovulatoryresponse with this compound. Kimura et al. [26] reported that a single injection of FSH in aluminum hydroxide gel waseffective in inducing superovulation in cattle, but aluminum hydroxide is commonly used as a vaccine adjuvant [3]which may preclude its use for superovulation. In another study, FSH dissolved in polyethylene glycol (PEG) wasreported to induce a satisfactory superovulatory response [18].

We have recently completed a series of experiments in which Folltropin-V diluted in a slow release formulation(SRF, Bioniche Animal Health) was administered in a single injection. In the first experiment [47], a single intramuscularinjection of Folltropin-V diluted in SRF (n = 29) was compared with the traditional twice-daily intramuscular injectionprotocol over 4 days (n = 29) in Red Angus donors. On Day 0 (beginning of treatment), all cows received a Cue-Mateand the P4 + E2 injection and on Day 4 treatments with 400 mg NIH-FSH-P1 Folltropin-V were initiated. Cows in Group1 received twice-daily intramuscular injections over 4 days and cows in Group 2 received a single intramuscularinjection in the neck. The single injection was prepared by diluting the Folltropin-V lyophilized powder in 1 mL of salinefor injection and then mixed with 9 mL of the SRF in the syringe, immediately before administration. In the AM and PMof Day 6, all cows received PGF

2á and Cue-Mates were removed in the PM. In the AM of Day 8, cows received 12.5

mg pLH and were FTAI 12 and 24 hours later. Ova/embryos were collected non-surgically on Day 15 and evaluated.Mean (± SEM) numbers of total ova/embryos, fertilized ova and transferable embryos did not differ (12.3 ± 1.5, 7.2± 1.1 and 4.9 ± 0.8 vs. 13.7 ± 2.1, 8.4 ± 1.4 and 6.4 ± 1.3; P>0.4) between twice daily and single injection groups,respectively. One cow in each treatment group had d” 2 CL at the time of ova/embryo collection, and one cow in thecontrol group and two cows in the single injection group did not produce any transferable embryos.

Additional experiments were conducted in several different breeds of donors to confirm the effectiveness ofthis protocol and to determine the appropriate dosage of Folltropin-V administered by the single intramuscular injection[41,47]. Overall, the single injection protocol resulted in a similar number of ova/embryos as the traditional twice-dailyFSH protocol. In Angus donors (138 superovulations), the number transferable embryos did not differ between 300 mgand 400 mg of Folltropin-V (6.1 ± 0.7 vs. 6.5 ± 0.7, respectively), but were higher than 200 mg (4.0 ± 0.5, P<0.02). InBrangus donors (69 superovulations), the number of transferable embryos did not differ between 260 mg and 300 mgof Folltropin-V (9.5 ± 1.6 vs 7.9 ± 1.5, respectively), but tended to be higher than 200 mg (5.2 ± 0.8, P<0.1). InBonsmara donors (64 superovulations), the number of transferable embryos did not differ between 200 mg (7.2 ± 0.8)and 300 mg (7.6 ± 1.0) of Folltropin-V.

Superovulation with a split administration of FSH

Although the single intramuscular injection of Folltropin-V diluted in SRF was highly efficacious in theinduction of superovulation in a variety of breeds of beef cattle, the optimum concentration of SRF was viscous andconsidered less practical to mix with Folltropin-V in the field. A 50% reduction in the concentration of SRF was muchless viscous and easier to mix with Folltropin-V, but a single intramuscular injection resulted in a lower superovulatoryresponse, presumably because more rapid absorption. Mean follicle sizes in the 50% SRF group were identical to theoriginal 100% SRF group on Days 4 (day of Folltropin-V injection), 5 and 6, but visibly less on Days 7 and 8, and largeron Days 10 and 11, suggesting that several follicles did not reach an ovulatory size and did not ovulate following

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estrus. We speculated that an additional injection of Folltropin-V in the 50% preparation 48 hours after initiatingtreatment would have caused follicles to continue growth to an ovulatory size as occurred in the 100% SRF group. Inthree studies involving 54 superovulations in Simmental, Brangus and Angus cross-bred cows, two intramuscularinjections of Folltropin-V in 50% SRF 48 hours apart induced a superovulatory response and ova embryo/embryoproduction that did not differ from twice daily intramuscular injections over 4 days. In addition, the numbers ofovulatory-sized follicles on the day of insemination did not differ among groups. The 50% SRF solution was mucheasier to mix with Folltropin-V than the previously used 100% SRF but it still took some time and manipulation to mixit properly. We speculated that an even more dilute preparation of the SRF may also be efficacious in a split intramuscularinjection protocol. Therefore, an experiment was designed to compare a split intramuscular injection of Folltropin-V in25% or 50% SRF with twice daily intramuscular injections in saline over 4 days. Twenty-nine beef cows (17 Angus and12 Simmental) were randomly assigned to one of three treatment groups to be superstimulated three times in a cross-over design. On Day 0 (random stages of the estrous cycle), cows received a Cue-Mate intravaginal device and anintramuscular injection of 5 mg estradiol-17â and 50 mg progesterone to synchronize follicle wave emergence. OnDay 4, superstimulatory treatments were initiated. Dilution of Folltropin-V lyophlized powder was done using 20 mL ofsaline for cows in the Control Group or 10 mL of 25% or 50% SRF solution for cows in the split injection groups. Allcows received a total dose of 300 mg Folltropin-V divided into twice daily intramuscular injections over 4 days(Control), or as an intramuscular injection of 200 mg (i.e. 67% of the total dosage) on Day 4 and 100 mg Folltropin-V(i.e. 33% of the total dosage) on Day 6 in 25% or 50% SRF, respectively. Prostaglandin was injected on Day 6 AM andPM and Cue-Mates were removed on Day 7 AM. Cows received 12.5 mg Lutropin-V on Day 8 AM and were inseminatedon Day 8 PM and 9 AM. Although the numbers of total ova/embryos collected were higher (P<0.05) in cows treatedwith the split injection treatments (25% SRF: 14.3 ± 2.1 and 50% SRF: 14.4 ± 2.0) than those in the Control Group(10.2 ± 1.8), there were no significant differences among groups in the number of fertilized ova and transferableembryos (Control: 6.7 ± 1.3 and 4.0 ± 0.8), Group 2 (25% SRF: 9.3 ± 1.9 and 6.1 ± 1.3) and Group 3 (50% SRF: 8.9± 1.4, and 5.0 ± 0.9). Data were interpreted to suggest that the split intramuscular administration of Folltropin-V dilutedin either 25% or 50% SRF results in a comparable superovulatory response to the traditional twice-daily intramuscularinjection protocol. The treatment schedule is depicted in Figure 2.

Figure 2. Treatment schedule used for superovulation of beef donor cows with a split injection of Folltropin-V in SRF. Donors receive aprogesterone-releasing device (Cue-Mate) along with 5 mg estradiol-17â and 50 mg progesterone im on Day 0. Superovulation is performedby injecting two-thirds of the total dosage of Folltropin-V (FSH) in SRF on Day 4 and the remaining one-third of the dosage on Day 6 AM.PGF2á is administered on Day 6 AM and PM and Cue-Mates are removed on Day 7 AM. Ovulation is induced with Lutropin-V (pLH) 24 hoursafter Cue-Mate removal (Day 8 AM), donors are fixed-time inseminated (AI) 12 and 24 hours later and ova/embryos are collected 7 daysafter pLH treatment.

V. CONCLUSIONS

The incorporation of protocols that control follicular dynamics and ovulation have the advantage of beingable to schedule the treatments quickly and without the need for detecting estrus in donor cows. These treatments arepractical and easy to perform by the farm staff, and more importantly, they do not depend on the skill and accuracyin estrus detection. However, E

2 which has been most useful in synchronizing follicle wave emergence is not available

in many countries around the world. Although the administration of GnRH to synchronize follicular wave emergenceyields variable results, pre-synchronization with a P4-releasing device improves the response to GnRH and allows for

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superovulation during the first follicular wave after ovulation, with results that did not differ from the use of E2. Recent

studies with the use of a proprietary slow-release formulation have shown that it is possible induce a consistentsuperovulatory response following administration of one or two injections of Folltropin-V, without adversely affectingthe number of transferable embryos.

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20 Demoustier J.M., Beckers J.F., Van Der Zwalmen P., Closset J., Gillard J. & Ectors F.R. 1988. Determination of porcine plasmaFolltropin-V levels during superovulation treatment in cows. Theriogenology. 30: 379-386.

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23 Edwards L.M., Rahe C.H., Griffin J.L., Wolfe D.F., Marple D.N. & Cummins K.A. 1987. Effect of transportation stress on ovarianfunction in superovulated Hereford heifers. Theriogenology. 28: 291–299.

24 Ginther O.J., Kastelic J.P. & Knopf L. 1989. Temporal associations among ovarian events in cattle during estrous cycles with two andthree follicular wave. Journal of Reproduction and Fertility. 87: 223-230.

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25 Hockley D.K., Bó G.A., Palasz A.T., Del Campo M.R. & Mapletoft R.J. 1992. Superovulation with a single subcutaneous injection ofFolltropin in the cow: Effect of dose and site of injection. Theriogenology. 37: 224.

26 Kimura K., Hirako M., Iwata H., Auki M., Kawaguchi M. & Seki M. 2007. Successful superovulation of cattle by a single administrationof FSH in aluminum hydroxide gel. Theriogenology. 68: 633-639.

26 Lane E.A., Austin E.J. & Crowe M.A. 2008. Estrus synchronisation in cattle-Current options following the EU regulations restricting useof estrogenic compounds in food-producing animals: A review. Animal Reproduction Science. 109: 1-16.

28 Laster D.B. 1972. Disappearance of and uptake of 125I FSH in the rat, rabbit, ewe and cow. Journal of Reproduction and Fertility. 30: 407-415.

29 Lindsell C.E., Murphy B.D. & Mapletoft R.J. 1986. Superovulatory end endocrine responses in heifers treated with FSH-P at differentstages of the estrous cycle. Theriogenology. 26: 209-219.

30 Looney C.R., Boutle B.W., Archibald L.F. & Godke R.A. 1981. Comparison of once daily FSH and twice daily FSH injections forsuperovulatin in beef cattle. Theriogenology. 15: 13-22.

31 Lovie M., García A., Hackett A. & Mapletoft R.J. 1994. The effect of dose schedule and route of administration on superovulatoryresponse to fooltropin in holstein cows. Theriogenology. 41: 241.

32 Macmillan K.L. & Thathcher W.W. 1991. Effect of an agonist of gonadotropin-releasing hormone on ovarian follicles in cattle. Biology ofReproduction. 45: 883-889.

33 Mapletoft R.J., Bennett-Steward K. & Adams G.P. 2002. Recent Advances in the superovulation of cattle. Reproduction, Nutrition andDevelopment. 42: 601-611.

34 Martinez M.F., Adams G.P., Bergfelt D., Kastelic J.P. & Mapletoft R.J. 1999. Effect of LH or GnRH on the dominant Follicle of the firstfollicular wave in weifers. Animal Reproduction Science. 57: 23-33.

35 Menchaca A., Pinczak A. & Rubianes E. 2002 Follicular recruitment and ovulatory response to FSH treatment initiated on Day 0 or Day3 postovulation in goats. Theriogenology. 58: 1713-1721.

36 Monniaux D., Chupin D., & Saumande J. 1983. Superovulatory responses of cattle. Theriogenology. 19: 55-81.

37 Murphy B.D. & Martinuk D. 1991. Equine Chorionic Gonadotropin. Endocrine Reviews. 12: 27-44.

38 Nasser L., Adams G.P., Bó G.A. & Mapletoft R.J. 1993. Ovarian superstimulatory response relative to follicular wave emergence inheifers. Theriogenology. 40: 713-724.

39 Nasser L.F., Bó G.A., Reis E.L., Menegati J.A., Marques M.O., Mapletoft R.J. & Baruselli P.S. 2003. Superovulatory responseduring the first follicular wave in Nelore Bos indicus donors. Theriogenology. 59: 530.

40 Pursley J.R., Mee M.O. & Wiltbank M.C. 1995. Synchonization of ovulation in dairy cows using PGF2á and GnRH. Theriogenology. 44:915-923.

41 Rogan D., Tríbulo A., Tríbulo H., Tríbulo R., Carballo D., Tríbulo P., Mapletoft R.J. & Bó G. A. 2010. Dose titration for superovulationof Brangus, Bonsmara and Braford donors with Folltropin-V by a single intramuscular injection. Reproduction, Fertility and Development.22: 365.

42 Schams D., Menzer D., Schalenberger E., Hoffman B., Hahn J. & Hahn R. 1977. Some studies of the pregnant mare serumgonadotrophin PMSG and on endocrine responses after application for superovulation in cattle. In: Sreenan J.M. (Ed). Control ofReproduction in the Cow. Martinus Nijhoff: The Hague, pp. 122-142.

43 Small J.A., Colazo M.G., Kastelic J.P. & Mapletoft R.J. 2009. Effects of progesterone presynchronization and eCG on pregnancyrates to GnRH-based, timed-AI in beef cattle. Theriogenology. 71: 698-706.

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45 Stoebel D.P., & Moberg G.P. 1982. Repeated acute stress during the follicular phase and luteinizing hormone surge of dairy heifers.Jounal of Dairy Science. 65: 92–6.

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47 Tríbulo A., Tríbulo H., Tríbulo R., Carballo D., Tríbulo P., Rogan D., Mapletoft R.J. & Bó G.A. 2010. Superovulation of angus donorswith a single intramuscular injection of Folltropin-V. Reproduction, Fertility and Development. 22: 367.

48 Walsh J.H., Mantovani R., Duby R.T., Overstrom E.W., Dobrinsky J.R., Enright W.J., Roche J.F. & Boland M.P. 1993. The effectsof once or twice daily injections of p-FSH on superovulatory response in heifers. Theriogenology. 40: 313-321.

49 Wock J.M., Lyle L.M. & Hockett M.E. 2008. Effect of gonadotropin-releasing hormone compared with estradiol-17â at the beginning ofa superovulation protocol on superovulatory response and embryo quality. Reproduction, Fertility and Development. 20: 228.

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Physiological Bases for Understanding Estrous Cycle DifferencesBetween Bos taurus and Bos indicus

Roberto Sartori1, Michele Ricieri Bastos2 & Maria Clara Costa Mattos2

ABSTRACT

Background: Although there is some information in the literature discussing differences of the estrous cycle of Bostaurus and Bos indicus cattle, most of the data derive from studies performed in temperate climate countries, underenvironmental and nutritional conditions very different than those found in tropical countries. Moreover, the physiologicalbasis for understanding the differences between Bos taurus and Bos indicus estrous cycles are still unknown. Thisreview explores the physiological and metabolic bases for understanding the key differences between the Bos taurusand Bos indicus estrous cycle. Moreover, it presents recent results of studies that have directly compared reproductivevariables between Zebu and European cattle.

Review: The knowledge of reproductive physiology, especially the differences between Bos taurus and Bos indicus,is important for the development and application of different techniques of reproductive management in cattle. In thisregard, overall, Bos indicus have a greater number of small ovarian follicles and ovulatory follicles are smaller ascompared to Bos taurus. Consequently, Zebu cattle also have smaller corpus luteum (CL). Nevertheless, circulatingconcentrations of steroid and metabolic hormones are not necessarily higher in European cattle. In fact, some studieshave shown that despite ovulating smaller follicles and having smaller CL, Bos indicus cows or heifers have highercirculating concentrations of estradiol, progesterone, insulin and IGF-I compared to Bos taurus females. In addition,there are also substantial differences between Bos indicus and Bos taurus cattle in relation to follicle size at the timeof selection of the dominant follicle.

Conclusion: Data from very recent studies performed in Brazil have corroborated results from previous reports thathave observed substantial differences in the estrous cycle variables of Bos indicus versus Bos taurus cattle. Thosedifferences are probably related to distinct metabolism and metabolic hormone concentrations between Zebu andEuropean cattle. This increased knowledge will allow for the establishment of more adequate reproductive managementprotocols in both breeds of cattle.

Keywords: reproductive physiology, Bos taurus, Bos indicus, steroid hormones, metabolic hormones, heat stress.

*Financial support from FAPESP of Brazil. 1Departamento de Zootecnia, Escola Superior de Agricultura “Luiz de Queiroz”, Universidadede São Paulo (USP), Av. Pádua Dias n.11, Bairro Agronomia, CEP 13418-900, Piracicaba, São Paulo, Brasil 2Departamento de ReproduçãoAnimal e Radiologia Veterinária, Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual Paulista “Júlio de Mesquita Filho”(UNESP), Distrito de Rubião Júnior s/n, CEP 18618-970, Botucatu, São Paulo, Brasil. CORRESPONDENCE: R. Sartori[[email protected] - Fax: + 55 (19) 3429-4007].

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I. INTRODUCTION

II. FOLLICLE RECRUITMENT AND DEVIATION

III. SIZE OF THE DOMINANT/OVULATORY FOLLICLE, CORPUS LUTEUM ANDREPRODUCTIVE HORMONES

IV. EFFECT OF METABOLIC HORMONES ON PRODUCTION AND METABOLISM OFSTEROID HORMONES

V. EFFECT OF HEAT STRESS ON CIRCULATING CONCENTRATIONS OF STEROIDSHORMONES

VI. CONCLUSIONS

I. INTRODUCTION

Many researchers have studied the reproductive physiology of Bos indicus and Bos taurus females. However,consistent information related to those genetic groups of cattle managed in tropical and subtropical environments stillare necessary because most of the data generated from those studies were produced under different conditions thanthose observed in a tropical environment, like Brazil. Furthermore, alterations in reproductive variables of Bos indicusand Bos taurus cattle may happen when nutritional and metabolic conditions of these animals change.

The purpose of this article is to discuss physiological and metabolic bases for understanding the differencesbetween the estrous cycle of Bos indicus and Bos taurus cattle.

II. FOLLICLE RECRUITMENT AND DEVIATION

Many studies have reported that the number of ovarian antral follicles is greater in Bos indicus than Bostaurus breeds [10,47]. Similarly, when comparing nonlactating Zebu and European cows under the same environmentaland nutritional conditions, Bastos et al. [6] observed a greater number of small follicles (2-5 mm) in the ovaries ofNelore (42.7 ± 5.9) than Holstein (19.7 ± 3.2) cows at the time of wave emergence. This difference in number of smallfollicles between those breeds persisted throughout the estrous cycle.

It is very likely that factors directly involved in the recruitment of small antral follicles are related to circulatingFSH concentrations and the Insulin/IGF-I system [54]. Recent studies have shown that Bos indicus females havelower circulating FSH [1], but higher plasma insulin [6] and IGF-I [1,50, M.R. Bastos, unpublished data] concentrationsthan Bos taurus cows or heifers.

The beginning of follicular growth and development through the preantral phase seems to be dependent ongrowth factors [54]. At that time, follicle development is gonadotropin-independent. Nevertheless, FSH receptormRNA was detected in follicles with only one or two granulosa layers (primary follicles), even before the formation oftheca interna [5,54]. In vivo and in vitro studies have shown that FSH at the preantral phase increases follicledevelopment, but is not essential for this phenomenon to occur. However, after recruitment (diameter e”4 mm),follicles become gonadotropin-dependent and growth factors are less relevant and exert a permissive role, sinceduring the antral phase follicles grow only up to 4 mm in diameter in the absence of gonadotropins [9,22,54].

Studies have demonstrated that IGF-I is important for the early stages of follicle development, becauseknockout of the IGF-I gene led to a severe impairment of preantral and early antral follicle growth in mice [reviewed by8]. Besides, changes in circulating insulin and IGF-I concentrations induced by nutrient management affect folliclerecruitment [54]. Moreover, small follicles in overfed heifers had a significant reduction in the expression of IGFBP-2and -4 mRNA, increasing the bioavailability of IGF-I. This is probably a critical factor for preantral follicle developmentalcontrol. In agreement, other studies have shown that most of the non-recruited follicles expressed IGFBP-2 mRNA[13,65], suggesting that follicles with IGFBP-2 mRNA expression are the ones that undergo atresia.

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In our recent study [6] we have observed that the plasma insulin concentrations before feed consumptionwere higher in the follicular phase as compared to the luteal phase in Nelore (11.9 ± 2.05 vs. 8.3 ± 1.47 µIU/mL) aswell as in Holstein (4.2 ± 1.05 vs. 2.2 ± 0.64 µIU/mL) cows. It is known that serum insulin concentrations changeduring the day, but these changes also occur during the estrous cycle, with a significant increase during the preovulatoryperiod [54]. Estradiol is a strong candidate to mediating these changes, because the increase in serum concentrationsof insulin occurs with the increase in circulating estradiol produced by the dominant follicle. It has been shown thatestradiol stimulates both the expression of insulin mRNA as well as its secretion by the pancreas cells [54].

Insulin is one of the main factors that control the release and bioavailability of IGF-I [2,21]. Therefore, wespeculate that higher concentrations of insulin, as well as IGF-I are responsible for the greatest number of smallfollicles in Bos indicus than Bos taurus cattle, especially because these metabolic hormones are increased during theperiovulatory period, when follicle recruitment occurs.

Another main difference between Bos indicus and Bos taurus is the diameter of the greatest growing follicleat the time of follicle deviation. Follicle deviation has been used to refer to the time at which differences in the growthrate between the future dominant and the future subordinate follicles become apparent [20]. The average diameter ofthe future dominant follicle at the time of deviation was 8.5 to 8.9 mm in Holstein females [6,20,45], whereas folliculardeviation occurred when the greatest growing follicle reached 5.4 to 6.2 mm in diameter in Nelore heifers [11,18,44]and 7.0 mm in diameter in nonlactating Nelore cows [6]. Despite those differences in follicle size, the time of theestrous cycle in which deviation occurs does not differ between Bos taurus and Bos indicus. On average, deviationoccurred between 2.3 and 2.8 days after wave emergence or ovulation in Holstein and Nelore females [6,11,18,20,44,45].This fact is only possible because follicle growth rate in Nelore (0.8 to 1.2 mm/day) [18] is lower than in Holstein (1.2to 1.6 mm/day) [45] cattle.

Studies [16,36] have shown that there is also a strong involvement of the IGF system in the selection ofthe dominant follicle. The synergism between IGF-I and FSH promotes estradiol synthesis in antral follicles. There isa negative association between estradiol concentration and low molecular weight IGFBP in follicular fluid [16,36] andthis association was already present on Day 2 of the first follicular wave in cattle [36]. At that moment, the diameterdifference between the dominant follicle and the largest subordinate follicle was only 1 mm. However, estradiolconcentration was four times greater and the levels of IGFBP-4 were 2.5 times lower in follicular fluid of the dominantfollicle than the largest subordinate one. According to that study, follicles with a greater responsiveness to FSH,acquire IGFBPase-4 earlier, increasing free IGF-I concentrations in follicular fluid which, in synergism with FSH,stimulates steroidogenesis increase. Then, estradiol exerts a negative feedback on FSH secretion, preventing otherfollicles of the same wave to acquire IGFBPases and become dominant.

As mentioned above, Zebu cattle have higher circulating concentrations of IGF-I and insulin than Europeancattle. It is believed that follicle diameter at deviation in Nelore is smaller than in Holstein cows and heifers, becausethese metabolic hormones increase the follicle responsiveness and sensitivity to FSH.

III. SIZE OF THE DOMINANT/OVULATORY FOLLICLE, CORPUS LUTEUM ANDREPRODUCTIVE HORMONES

Studies have demonstrated that the maximum size of the dominant follicle and CL are greater in Bos taurusthan Bos indicus breeds. For example, the maximum diameter of the ovulatory follicle were, on average 14 to 17 mmin Holsteins [19,46] and 11.3 to 12.3 mm in Nelore cattle [15,44]. Similarly, the maximum CL diameter was between20 and 30 mm in Bos taurus [19,46] and between 17 to 21 mm in Bos indicus [15,35,47] females.

Despite those differences in size favoring European cattle, Zebu have greater circulating concentrations ofsteroid and metabolic hormones than Bos taurus cattle. In relation to circulating estradiol, studies that have directlycompared Bos taurus and Bos indicus are scarce. In one of those studies [1], circulating estradiol peak concentrations(8.9 ± 1.6 vs. 9.1 ± 1.4 vs. 8.7 ± 1.4 pg/mL in Brahman, Angus and Senepol cows, respectively) did not differ amongbreeds. However, this is the only study that we are aware of that have observed a greater diameter of the ovulatoryfollicle in Bos indicus than in Bos taurus cattle (15.6 ± 0.5 vs. 12.8 ± 0.4 vs. 13.6 ± 0.4 mm in Brahman, Angus andSenepol cows, respectively).

According to Randel [33], Bos indicus females and crossbreds have lower progesterone concentration pergram of luteal tissue than Bos taurus cattle. However, Segerson et al. [47] found no difference in progesterone

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production by the CL between Brahman and Angus cows (75.8 ± 11.3 and 65.9 ± 5.3 mg progesterone/g of CL,respectively; P > 0.10). In contrast, studies performed in Brazil did not corroborate those reported above. A study withBos indicus (Nelore and Gir), Bos taurus (Angus and Holstein) and Bos indicus x Bos taurus (Angus x Nelore and Girx Holstein) heifers has detected higher circulating progesterone concentrations in Bos indicus females during thetime in which they received an intravaginal progesterone-releasing device [10]. Similarly, although in our recent study[6], the maximum diameter of the ovulatory follicle (15.8 ± 1.4 vs. 13.4 ± 1.4mm) as well as CL volume on Day 14 ofthe estrous cycle (7890.90 ± 1752.40 vs. 4916.64 ± 1733.40 mm3) were greater in Holstein cows, plasma estradiol,progesterone, insulin and IGF-I concentrations were higher in Nelore cows.

In relation to higher circulating progesterone concentration, the authors [6,10] speculate that this findingmay be due to a higher liver metabolism of this steroid hormone in European as compared to Zebu cattle. It is knownthat the liver metabolism of steroid hormones is correlated with the dry matter intake [42,53]. However, in our study[6], all cows were fed the same maintenance diet and dry matter intake per kg of body weight was 1.37% for Neloreand 1.54% for Holstein cows, according to NRC (2000) recommendation. Therefore, based on these results, wespeculate that a possible higher steroid metabolism in Holstein cows may be inherent to the breed and not only aconsequence of higher feed intake. Moreover, Nelore cows may also have lower hepatic metabolism and/or increasedsteroid production by the ovaries than Holstein cows. Finally, heat stress can be another important factor related tolower circulating estradiol and progesterone in Holstein cows. These hypotheses will be discussed in more detail atthe following sessions.

IV. EFFECT OF METABOLIC HORMONES ON PRODUCTION AND METABOLISM OFSTEROID HORMONES

It is speculated that Bos indicus have lower steroid metabolism than Bos taurus females and this may bedue to the higher circulating insulin in Zebu cattle. The greatest amount of circulating progesterone is inactivated orcatabolized in hepatocytes by cytochrome P450 2C (CYP2C) and cytochrome P450 3A (CYP3A) enzymes. The mainmetabolites are 21-hydroxyprogesterone and 6â-hydroxyprogesterone, respectively [30,31]. Some studies have shownthat insulin alters the expression of these enzymes. Studies [41,48] in which hepatocytes of rodents were cultured invitro, observed a decrease in expression of CYP3A mRNA when physiological doses of insulin were added to themedia. They also observed an insulin dose-dependent decrease in 6â-hydroxyprogesterone. Recently, Lemley et al.[28] showed that insulin alters the expression of these enzymes in dairy cows. These authors first induced theincrease of insulin by propylene glycol infusion and detected lower expression of CYP3A. Subsequently, insulin andglucose were infused to promote a hyperinsulinemic-euglycemic curve. It was observed that insulin caused a dose-dependent decrease in expression of both CYP2C and CYP3A enzymes.

Moreover, results from recent studies in which Zebu have higher circulating insulin and IGF-I concentrationsthan European cattle, allow us to hypothesize that Nelore cows or heifers have a higher steroid production and lowersteroid metabolism than Holstein females. Both insulin and IGF-I act as potent stimulators of granulosa cells proliferationand steroidogenesis in cattle [54]. IGF-I acts in synergism with FSH on steroidogenesis by increasing the P450aromatase activity [14]. In vitro culture of bovine granulosa cells with 100 ng/ml of insulin stimulated mRNA expressionand P450 aromatase activity, and increased estradiol secretion by these cells [49].

Recently, bovine granulosa cells were cultured under different concentrations of IGF-I (1, 50 and 100 ng/mL) in a serum-free system without insulin [29]. The authors observed that cells cultured with IGF-I (50 or 100 ng/mL)had a significant increase in 17β-estradiol production, in cell number, in mRNA expression of genes related tosteroidogenesis (CYP11A1, HSD3B1, and CYP19A1) and of genes that encode receptors for IGF-I and FSH (FSHRand IGF-IR). Cells cultured only with FSH did not have any significant effect. Besides, it was reported that CL alsohas IGF-I receptors, and that IGF-I may increase gonadotropin activity and progesterone synthesis [43].

Another important action of insulin was shown by Armstrong et al. [2]. The authors found that highercirculating insulin concentrations induced by high feed intake decreased IGFBPs expression, increasing thebioavailability of IGF. It was also cited that nutritional status, i.e., higher circulating insulin, can alter the amount andtype of circulating IGFBPs [21]. Lower circulating IGFBPs, may indicate higher free IGF-I concentration.

Thus, based on the data presented above, we must consider the possibility that steroid hormones productionin follicles and CL is, in fact, greater in Bos indicus than in Bos taurus, and insulin and IGF-I may be responsible for

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that. However, to date, there are no reports that have evaluated and directly compared steroid production and metabolismin Bos taurus vs Bos indicus females.

V. EFFECT OF HEAT STRESS ON CIRCULATING CONCENTRATIONS OF STEROIDSHORMONES

Heat stress during summer substantially contributes to lower fertility and production, especially in Bostaurus breeds. In contrast, Bos indicus breeds are known for their adaptation to adverse conditions at tropicalenvironments, especially due to their higher thermo tolerance as compared to European breeds. However, the impactof heat stress on Zebu breeds should not be ignored [25,51].

The effects of environmental temperature on animal performance can be measured by establishing acritical temperature [7] or through an index of equivalent temperature, which incorporates temperature and humidity ofthe air (THI) [32].

According to the literature, milk production and dry matter intake decrease when the maximum THI reaches77. Subsequent researches have determined maximum, intermediate and minimum THI of 76, 72 and 64, respectively,for Bos taurus females. In fact, THI can be used as an estimative for predicting the effects of heat stress on cattleperformance [reviewed by 55].

In relation to the heat stress influence on neuroendocrine and reproductive physiology of Bos taurus andBos indicus, few studies have reported that FSH plasma concentrations are increased in cows under heat stress,probably because circulating inhibin concentrations are reduced by the impairment of follicular development [34].

Data regarding plasma LH concentrations in animals under heat stress are controversial. Studies haveshown that cows under heat stress have reduced amplitude and frequency of LH pulses. However, the preovulatoryLH peak was reduced in heifers but not in cows [reviewed by 34]. It is suggested that these differences are related tothe preovulatory circulating estradiol concentrations found in these studies, in which cows with lower plasma estradiolconcentrations had less LH pulses, or vice-versa [17]. The growth of the dominant follicle is regulated by LH pulsefrequency. If heat stress decreases LH pulses, there is an impairment in follicle development, and consequently on itsestradiol production and secretion. In fact, several studies have shown that plasma estradiol concentrations arereduced in dairy cows or heifers under heat stress [17,40,56,57,60,63]. According to the majority of the studies thathave reported reduced LH concentrations in animals under heat stress, the dominant follicle develops at low LHconcentrations, resulting in decreased estradiol secretion, what directly affects estrus expression. In this case, evenhigh FSH concentrations are not able to overcome the deleterious effect of the LH decrease on estradiol synthesis bythe preovulatory follicle. Moreover, the decrease in steroidogenic capacity of follicles in cows or heifers under heatstress may be related to lower activity of aromatase in granulosa cells that has been reported, decreasing estradiolconcentration in follicular fluid of dominant follicles [3].

A seasonal study has evaluated the function of dominant follicles on Day 7 of the estrous cycle. Estradiolconcentrations in follicular fluid were lower during the summer. This decrease was due to a drastic reduction in theproduction of androstenedione by theca cells during summer (4.1 ± 0.5 and 1.1 ± 0.3 ng/105 of viable cells for winterand summer, respectively). Thus, the production of estradiol by granulosa cells decreased around 50% during thewarmer months (12.6 ± 1.7 and 6.6 ± 0.9 ng/105 of viable cells for winter and summer, respectively) [60]. Moreover,the percentage of viable granulosa cells from follicles during the summer decreased ~60%, contributing to thereduced blood estradiol concentration [61]. Theca cells incubated at elevated temperatures or from follicles collectedin the winter of cows under acute heat stress in chambers have also shown reduced androstenedione production [60].

In contrast to other studies, no difference in serum or follicular concentrations of estradiol and progesteronewas detected in lactating [12] or nonlactating [23] Holstein cows under acute heat stress and the control group.

The effect of heat stress on blood progesterone concentration is even more controversial. Studies havereported that circulating progesterone concentrations were increased, not influenced or decreased (majority of thestudies) by heat stress (Table 1). Two of these studies did not detect effect of heat stress on plasma progesteroneconcentrations [23,24]. However, in another study, progesterone concentrations increased after Day 16 of the estrouscycle and luteolysis was delayed in lactating cows under heat stress (29.1 ± 2.4 vs. 20.4 ± 2.4 days) [56]. Thecontroversial results reported in the literature, may be due to interference of many uncontrolled factors, such as: typeof heat stress (acute or chronic), dry matter intake, liver metabolism, age and stage of lactation [61].

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Table 1. Influence of acute or chronic heat stress on plasma progesterone concentration. Adapted from Rensis& Scaramuzzi [34] and Wolfenson et al. [62].

Reference Progesterone concentration Type of stress

[52,56] Increased Acute (warming chambers)

[23,24] Not altered Acute (warming chambers)

[37] Increased Acute (solar radiation)

[39] Not altered Acute (solar radiation)

[27,38,51,58,59,64] Decreased Chronic

Most of the authors suggest that plasma progesterone concentrations are reduced in animals under chronicheat stress, typically observed in natural conditions of summer. However, under acute heat stress, such as exposureto direct solar radiation or use of warming chambers, increases in circulating progesterone have been reported[26,62]. This higher progesterone concentration in cows under acute heat stress may be related to a higher progesteronesecretion by the adrenal gland [26], and not necessarily by the CL. In vitro studies support this hypothesis, becauseprogesterone production by luteinized theca cells obtained from cows during summer was lower than during winter[62].

The study performed by Alvarez et al. [1] that was mentioned previously, in which Brahman cows hadgreater follicles and CL than Angus cows, was performed during summer in Florida. These unexpected resultspossibly reflect a more pronounced effect of heat stress on follicular dynamics of Angus (Bos taurus) than Brahman(Bos indicus) cows. However, those differences in size between breeds were not reproduced on circulating steroidconcentrations, since there was no difference in plasma estradiol or progesterone concentrations between Brahmanand Angus cows.

A study performed in Brazil [51] found no immediate effect of heat stress on reproductive function of Gircows, possibly due to the higher thermo tolerance and rusticity of Zebu breeds. However, the extended heat stressthose cows underwent promoted deleterious effects on follicular growth by increasing the number and diameter oflarge follicles, through an ineffective mechanism of selection and follicle dominance. Indeed, Sartori et al. [46]observed that lactating dairy cows during summer, had a higher incidence of follicular codominance than heifers,resulting in a higher percentage of multiple ovulations. Delayed ovulation or failure to ovulate was also more frequentin lactating cows than in nulliparous heifers.

In our recent study, performed during the summer of 2010 in Brazil, the maximum diameter of the ovulatoryfollicle, as well as the CL volume was greater in nonlactating Holstein than Nelore cows, as mentioned above [6].However, plasma progesterone concentrations on Day 7 and 14 of the estrous cycle and peak estradiol were higherin Bos indicus than Bos taurus cows. Besides potential differences in steroid metabolism between breeds, as discussedbefore, it is possible that estradiol and progesterone production and secretion by the ovaries of Holstein cows mayhave been negatively influenced by heat stress, given that the maximum and minimum THI recorded were 87.5 and64.5, respectively. Probably, although THI was high during that period of study, Nelore cows may still have been inthermal comfort zone due to their greater adaptation to the tropical environment, differently than Holstein cows.

Finally, the mechanisms involved in gonadotropins and steroid hormones secretion in animals under heatstress are poorly understood. However, it seems that the main action of heat stress is at the hypothalamic-pituitary-gonadal axis, decreasing GnRH and LH release and, consequently, estradiol production by the preovulatory follicle.Moreover, plasma progesterone concentrations are influenced by multifactorial factors; being the reason why datapresented in the literature are so controversial.

VI. CONCLUSIONS

After extensive literature review, it was observed that there are still few studies that have effectively anddirectly compared the reproductive physiology of Bos taurus and Bos indicus subspecies. Some information regardingthe differences in the estrous cycle are known, however, the vast majority of the work was not developed in the

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tropical climate conditions found in Brazil. In this regard, very recent studies performed in Brazil with direct comparisonsbetween Zebu and European cattle have provided important facts that can be used to optimize reproductivemanagement and enable the development of specific biotechnologies for Bos taurus or Bos indicus cattle.

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Use of LH or eCG in the Last Day of Superestimulatory Treatment

R.L. Ereno1, F.S. Rosa1, A.C.S. Oliveira2, M.F.G. Nogueira3 & C.M. Barros1a

ABSTRACT

In the last decades several hormonal treatments to induce multiple ovulation and embryo transfer (MOET) have beendeveloped. Tight control of the time of ovulation allowed the use of fixed-time artificial insemination (FTAI) in embryosdonors, facilitating animal management. Although, protocols that allow FTAI have evolved and yield as much embryoas conventional protocols that requires estrus detection, substantial increase in viable embryo production has notbeen observed in superestimulated bovine cattle. The present mini-review put emphasis on superstimulatory protocolsin which the last two doses of pFSH are replaced by eCG or LH. Recent results indicate that an extra LH stimulus(using eCG or LH), on the last day of P-36 superestimulatory treatment, seems to improve transferable embryo yieldin both Bos taurus and Bos indicus cattle.

Keywords: embryo transfer, superovulation, bovine, FSH, LH, eCG.

1Departamento de Farmacologia, Instituto de Biociências de Botucatu, 2Departamento de Reprodução Animal, FMVZ. 3Departamento deCiências Biológicas, Faculdade de Ciências e Letras de Assis. Universidade Estadual Paulista (UNESP), São Paulo, Brasil.CORRESPONDENCE: R.L. Ereno [[email protected]]. Parte desta mini-revisão foi apresentada na reunião anual da IETS/SBTE(Janeiro de 2010, Córdoba, Argentina).

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I. INTRODUCTION

Bovine females have been the subject of several researches aiming to improve exploitation of gametes.Gordon [19] reported that offspring calves posses more than 100.000 oocytes in their ovaries, which will generate0.01% of viable products, i.e., 10 descendents during their reproductive lives. Along with artificial insemination,multiple ovulation and embryo transfer (MOET) and in vitro production of embryos are useful tools to increasegametes production [25].

Several treatments to induce multiple ovulations in cattle have been recommended [4,3,6,10]. Equinechorionic gonadotropin administered alone [13] or in combination with eCG antiserum [14], FSH from swine, ovine orequine pituitary extracts [16], or recombinant bovine FSH [22], are some of the hormones that have been utilized toinduce superovulation in the cow.

It has been reported that the presence of a dominant follicle at the beginning of the superstimulatorytreatment decreases embryo production [18,23]. A few strategies have been developed to avoid the presence of thedominant follicle when initiating superstimulatory treatments, such as starting FSH on the first day of the estrouscycle [20,34], aspirating the dominant follicle or all follicles larger than 5 mm in diameter [8], or synchronizing thefollicular wave with estradiol and progesterone [9,11].

Bo et al. [9,11] have shown that the use of progesterone in association with intramuscular administration ofestradiol promotes follicle atresia and induces emergence of a new follicle wave approximately 4 days after treatment.To avoid the presence of the dominant follicle, the FSH superstimulatory treatment is initiated at the beginning of thenew follicle wave i.e., 4 days after insertion of the progesterone intravaginal device and administration of estradiol.Two days after the first FSH treatment, a luteolytic dose of PGF2α is administered and 12 hours later the intravaginaldevice is removed. Donor cows are artificially inseminated 12 and 24 hours after estrus detection. Seven days later,the embryos are collected, classified and cryopreserved or transferred.

It has been suggested that follicles that did not ovulate following FSH superstimulation did not developnormally, or did not possess sufficient LHR to be activated by the preovulatory LH surge [39,15,21]. Thus, it has beenproposed that postponing the preovulatory LH surge may be a method of increasing embryo production [15,21], andallowing for FTAI in superstimulatory protocols [4,5,6].

Barros & Nogueira [4] examined the efficacy of different protocols in which the expected time of ovulationwas postponed by 6 to 12 hours and ovulation was induced by administration of LH or GnRH [4,28]. Although theseprotocols did not increase the quantity of viable embryos significantly when compared to estrus detection protocols,it was possible to control the ovulation time with the hormonal treatments, allowing the use of FTAI. From theseexperiments, a new protocol was developed, called the P-36 protocol [5]. In this protocol, the intravaginal progesteronereleasing device was left in place for up to 36 hours after PGF2α administration (that is why the protocol was calledP-36) and ovulation was induced with exogenous LH, administered 12 hours after withdrawal of the progesteronedevice (i.e., 48 hours after PGF2á administration). Since ovulation occurs between 24 and 36 hours after LHadministration [23], FTAI was scheduled for 12 and 24 hours after LH, avoiding the necessity of estrus detection. Theeffectiveness of the P-36 protocol has been confirmed [4-6], and more recently, an average of 13.3±0.75 totalstructures and 9.4±0.63 viable embryos, with a viability rate of 71.0% (1279/1807) following 136 embryo collectionsin Nelore cows has been reported [30]. These results were comparable to those reported in studies in which Nelorecows were inseminated 12 and 24 hours after onset of behavioral estrus [27,28].

Protocol P-24, a variation of the P-36 protocol in which the progesterone device is removed 24 hours afterPGF2a and LH is administered 24 hours later (48 hours after PGF2a), has been utilized in Nelore females withcomparable results to those obtained with P-36 protocol [6,40].

Although donors are typically inseminated twice, 12 and 24 hours after administration of LH or GnRH in theP-36 protocols [5,28], it is possible to use a single FTAI. The use of a single straw semen did not significantly reducethe viability rate when FTAI was scheduled at 16 hours after LH administration (viability rates of 52.4 and 58.3%,respectively; [6]) for one vs two FTAI in the P-36 protocol. Nevertheless, special attention must be paid to both qualityand quantity of semen that is used for a single FTAI in superstimulated donors, in order to avoid a decline in viableembryo production [5,30].

Another modification of the P-36 protocol has been examined in Bonsmara [2] and Nelore cattle [1], inwhich the last two FSH doses were replaced by administration of eCG. It was expected that on the last day of the

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superstimulatory treatment protocol most of the follicles had already acquired LH receptors [17] and would grow inresponse to eCG due to its capacity to stimulate both LH and FSH receptors [26]. As a consequence, thissuperestimulatory protocol with eCG would increase ovulation rate and embryo production. Nelore cows (n = 20) wererandomly allocated to two groups: P-36 protocol and P-36/eCG protocol, and each animal received both treatments ina crossover experimental design. The animals in the P-36 group received the “original” P-36 treatment protocol, whilethose in the P-36/eCG group had the last two pFSH treatments replaced by two 200 IU doses of eCG (i.m.). Thenumber of follicles with a diameter >6 mm at the time of administration of pLH (21.1 ± 2.9 vs 15.3 ± 2.1) and of thetotal number of embryos/ova recovered (10.0 ± 1.5 vs 6.7 ± 1.2, P < 0.03) were greater in animals that received eCG[1]. These results indicate that the administration of eCG stimulated final follicle growth, possibly due to the presenceof LH receptors in follicles larger than 7 mm [31,37], resulting in a greater number of follicles capable of ovulating inresponse to the administration of exogenous LH. Consequently, eCG increased the total number of ova/embryosrecovered, but there was no significant increase in the mean number of viable embryos from cows treated with eCG(7.3 ± 1.20 vs 5.1 ± 1.10); however, the total number of viable embryos produced by cows treated with the P-36/eCGprotocol (146 vs102) suggests that it is advantageous to replace the last two doses of pFSH with eCG. Similarpositive results were recently obtained in 47 Brangus females using P-36/eCG (10.9 ± 1.5 viable embryos) ascompared to P-36 (7.1 ± 1.4; [33]). On the other hand, Sartori et al. [36] reported that there was no increase on embryoproduction after using eCG in a P-24 protocol (3.7 ± 0.5 viable embryos) as compared to the P-24 protocol alone (4.9± 0.7) in Nelore heifers. However, the same authors have recently found in Sindhi females that adding eCG to the P-36 protocol increased the number of viable embryos (6.5 ± 1.2 vs 2.4 ± 0.7; P < 0.01; [24]). These data indicate thatadding eCG to the P-36 protocols seems to be advantageous, and its use on the P-24 protocol needs to be furtherinvestigated. The difference between these two protocols is that in the P-24 protocol the progesterone device isremoved 12 hours earlier than in the P-36 protocol, and, consequently, may allow earlier LH secretion that couldadvance oocyte maturation and disrupt fertilization following FTAI.

Taking into account that consecutive use of eCG reduces bovine embryo production [7], presumably due toantibody production against eCG, replacement of eCG by LH in the last day of superestimulatory treatment, wasrecently investigated. Rosa et al. [35] studied embryo yield in Angus cows (n = 22) treated with P-36/LH60 (controlgroup), P36/eCG (the last two FSH doses were replaced by two 200 IU doses of eCG), P36/LH (the last two FSHdoses were replaced by two 1.0 mg doses of LH), and P36/FSH+LH (the last two FSH doses were immediatelyfollowed by two 1.0 mg doses of LH). They observed that replacement of eCG by LH (group P36/LH), resulted in adecline (P < 0.05) in transferable embryos, when compared to the others groups. However, addition of LH to the lasttwo doses of pFSH (P36/FSH+LH) improved embryo quality and numerically increased the total embryo yield (87)when compared to Control (43) and P36/LH group (13), and was similar to P36/eCG (67). These results indicate thateCG can not be replaced by LH (2.0 mg) in Angus cows. However, FSH+LH may be used instead of eCG, in the lastday of the superestimulatory treatment.

In order to test if a higher dose of LH would have beneficial effect on embryo production, Oliveira et al. [32],allotted Nelore cows (n = 25) in 4 groups: P-36, P-36/eCG (the last two FSH doses were replaced by two 200 IU dosesof eCG), P36/LH2.0 (the last two FSH doses were replaced by two 1.0 mg doses of LH), and P-36/LH4.0 (the last twoFSH doses were replaced by two 2.0 mg doses of LH). Each donor was superestimulated four times and received alltreatments (crossover). In spite of the tendency of lower ovulation rate in LH2.0 as compared to eCG group (50.6 vs67.8%, respectively, P = 0.06), there was no significant difference among treatments P-36 (3.3 ± 0.7), P-36/eCG (4.5± 0.5), P-36/LH2.0 (3.7 ± 0.8) and P-36/LH4 (4.2 ± 1.0) for viable embryo yield. It was concluded that eCG can bereplaced by pLH (4.0 mg), in the last day of superestimulatory treatment of P-36 protocol, without affecting viableembryos yield in Nelore cows. Overall, these experiments indicate that an extra LH stimulus (using eCG or LH), on thelast day of P-36 superestimulatory treatment, seems to improve transferable embryo yield in both Bos taurus andBos indicus cattle. Additional experiments are warranted to confirm and amplify these findings.

Acknowledgments. The authors are grateful to FAPESP (Sao Paulo – Brazil) for funding, and CAPES (Brasilia –Brazil) and FAPESP for fellowships to graduate students.

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4 Barros C.M. & Nogueira M.F.G. 2001. Embryo transfer in Bos indicus cattle. Theriogenology. 56: 1483-1496 .

5 Barros C.M. & Nogueira M.F.G. 2005. Superovulation in zebu cattle: protocol P-36. Embryo Transfer Newsletter. 23: 5-9.

6 Baruselli P.S., De Sá Filho M.F., Martins C.M., Nasser L.F., Nogueira M.F.G., Barros C.M. & Bó G.A. 2006. Superovulation andembryo transfer in Bos indicus cattle. Theriogenology. 65: 77-88.

7 Baruselli P.S., Martins C.M., Sales J.N.S. & Ferreira R.M. 2008. Novos avanços na superovulação de bovinos. Acta ScientiaeVeterinariae. 36: 433-448.

8 Bergfelt D.R., Lightfoot K.C. & Adams G.P. 1994. Ovarian synchronization following ultrasound-guided transvaginal follicle ablation inheifers. Theriogenology. 42: 895-907.

9 Bó G.A., Adams G.P., Caccia M., Martinez M., Pierson R.A. & Mapletoft R.J. 1995. Ovarian follicular wave emergence after treatmentwith progestogen and estradiol in cattle. Animal Reprodution Science. 39: 193–204.

10 Bó G.A., Baruselli P.S., Chesta P.M. & Martins C.M. 2006. The timing of ovulation and insemination schedules in superstimulated cattle.Theriogenology. 65: 89-101.

11 Bó G.A., Baruselli P.S. & Martinez M.F. 2003. Pattern and manipulation of follicular development in Bos indicus cattle. AnimalReprodution Science. 78: 307–326.

12 Bodensteiner K.J., Wiltbank M.C., Bergfelt D.R. & Ginther O.J. 1996. Alterations in follicular estradiol and gonadotropin receptorsduring development of bovine antral follicles. Theriogenology. 45: 499-512.

13 Boland M.P., Crosby T.F. & Gordon I. 1978. Morphological normality of cattle embryos following superovulation using PMSG.Theriogenology. 10: 175.

14 Dieleman S.J., Bevers M.M. & Gielen J.T.H. 1987. Increase of the number of ovulations in PMSG/PG-treated cows by administrationof monoclonal anti-PMSG shortly after the endogenous LH peak. Theriogenology. 27: 222.

15 D’Occhio M.J., Sudha G., Jillela D., White T., Maclellan L.J., Whlsh J., Trigg T.E. & Miller D. 1997. Use of GnRH agonist to preventthe endogenous LH surge and injection of exogenous LH to induce ovulation in heifers superstimulated with FSH: a new model forsuperovulation. Theriogenology. 47: 601-613.

16 Donaldson L.E. 1989. Porcine, equine and ovine FSH in the superovulation of cattle. Theriogenology. 31:183 .

17 Fernandes P. 2008. Expressão dos transcritos do receptor de LH em células da granulosa de fêmeas Nelore submetidas ou não àsuperestimulação ovariana. 71f. Botucatu, SP. Tese (Doutorado em Medicina Veterinária) – Programa de Pós-Graduação em MedicinaVeterinária, Universidade Estadual Paulista, Botucatu.

18 Giubault L.A., Grasso F., Lussier J.G., Roullier P. & Matton P. 1991. Decreased superovulatory response in heifers superovulatedin the presence of a dominant follicle. Journal of Reprodudction and Fertility. 91: 89.

19 Gordon I.R. 2003. (Ed) Laboratory production of cattle embryos. 2.ed. London: CABI publishing, pp.548.

20 Goulding D., Williams D.H., Duffy P., Boland M.P. & Roche J.F. 1990. Superovulation in heifers given FSH initiated either at day 2 orday 10 of the estrous cycle. Theriogenology. 34: 767-778.

21 Liu J. & Sirois J. 1998. Follicle size-dependent induction of prostaglandin G/H synthase-2 during superovulation in cattle. Biology ofReproduvtion. 58: 1527-1532

22 Looney C.R. & Bondioli K.R. 1988. Bovine FSH produced by recombinant DNA technology. Theriogenology. 29: 235.

23 Lussier J.P., Lamothe P. & Pacholek X. 1995. Effects of follicular dominance and different gonadotrophin preparations on thesuperovulatory response in cows. Theriogenology. 43: 270.

24 Mattos M.C.C., Bastos M.R., Guardieiro M.M., Carvalho J.O., Mourão G.B., Barros C.M. & Sartori R. 2010. Improvement of embryoquality by the replacement of the last tow doses of porcine follicle-stimulating hormone by equine gonadotropin in superstimulated Sindidonors. (abstract) Reproduction Fertility and Development. 22: 364.

25 Merton J.S., Roos A.P.W., Mullaart E., Ruigh L., Kaal L., Vos P.L.A.M. & Dieleman S.J. 2007. Factors affecting oocyte quality incommercial application of embryo technologies in the cattle breeding industry. Theriogenology. 67: 1233-1238.

26 Murphy B.D. & Martinuk S.D. 1991. Equine chorionic gonadotrophin. Endocrine Reviews. 12: 27-44.

27 Nogueira M.F.G. & Barros C.M. 2003. Timing of ovulation in Nelore cows superstimulated with P36 protocol. (resumo) Acta ScientiaeVeterinariae. 31: 509.

28 Nogueira M.F.G., Barros B.J.P., Teixeira A.B., Trinca L.A., D’Occhio M.J. & Barros C.M. 2002. Embryo recovery and pregnancyrates after the delay of ovulation and fixed time insemination in superstimulated beef cows. Theriogenology. 57: 1625-634.

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29 Nogueira M.F.G., Buratini Jr.J. & Barros C.M. 2005. Tratamento com hCG e LH, como indutores da ovulação em doadorassuperestimuladas da raça Nelore, não altera a produção embrionária ou a taxa de prenhez. Acta Scientiae Veterinariae. 33: 263.

30 Nogueira M.F.G., Buratini Jr.J., Price A.C.S., Castilho M.G.L. & Barros C.M. 2007. Expression of LH receptor mRNA splice variantsin bovine granulosa cells: Changes with follicle size and regulation by FSH in vitro. Molecular Reproduction and Development. 74: 680-686.

31 Nogueira M.F.G., Fragnito P.S., Trinca L.A. & Barros C.M. 2007. The effect of type of vaginal insert and dose of pLH on embryoproduction, following fixed-time AI in a progestin-based superestimulatory protocol in Nelore cattle. Theriogenology. 67: 655-660.

32 Oliveira A.C.S., Mattos M.C.C., Bastos M.R., Lunardi L.H., Surjus R.S., Sartori R. & Barros C.M. 2010. Eficiência do protocolosuperestimulatório P-36, associado à administração de eCG ou LH, em vacas da raça Nelore (resumo). Acta Scientiae Veterinariae. 38.

33 Reano I., Carballo D., Tribulo A., Tribulo P., Balla E. & Bó G.A. 2009. Efecto de la adicion de eCG a los tratamientos superovulatorioscon Folltropin-V en la produccion de embriones de donantes de embriones. (resumo) VIII Simposio internacional de reproduccionanimal – IRAC. 1: 54.

34 Roberts A.J., Grizzle J.M. & Echternkamp S.E. 1994. Follicular development and superovulation response in cows administredmultiple FSH injections early in the estrous cycle. Theriogenology. 42: 917-929.

35 Rosa F.S., Bonotto A.L.M., Trinca L.A., Nogueira M.G.F. & Barros C.M. 2010. Eficiência do protocolo superestimulatório P-36,associado a administração de eCG ou LH em doadoras da raça Angus (resumo). Acta Scientiae Veterinariae. 37p.

36 Sartori R., Guardieiro M.M., Barros C.M., Bastos M.R., Machado G.M., Leme L.O. & Rumpf R. 2008. Lack of improvement onembryo production by the replacement of the last two doses of pFSH by eCG in superestimulated Nelore hefeirs. (abstract) Reproduction,Fertility and Development. 21: 245-246.

37 Simões R.A.L., Rosa F.S., Piagentini M., Castilho A.C.S., Ereno R.L., Nogueira M.F.G., Buratini J. & Barros C.M. 2010. Folliculardiameter, ovulation rate, and LH receptor gene expression in Nelore cows (abstract). Reprodution, Fertiliti and Development. 22: 270.

38 Van de Leemput E.E., Vos P.L.A.M. Hyttel P., Van den Hurk R., Bevers M.M., Van der Weijden G.C. & Dieleman S.J. 2001. Effectsof brief postponement of the preovulatory LH surge on ovulation rates and embryo formation in eCG/prostaglandin-treated heifers.Theriogenology. 55: 573-592.

39 Xu Z., Garverick H.A., Smith G.W., Smith M.F., Hamilton S.A. & Youngquist R.S. 1995. Expression of follicle-stimulating hormoneand luteinizing hormone receptor messenger ribonucleic acids in bovine follicles during the first follicular wave. Biology of Reproduction.53: 951-957.

40 Zanenga C.A., Pedrosa M.F., Lima G.F., Marques M.O., Santos I.C.C., Valentim R. & Baruselli P.S. 2003. Comparação entre doisprotocolos de superovulação com inseminação artificial em tempo fixo em vacas Nelore (Bos taurus indicus). Acta Scientiae Veterinariae.31: 626-627.


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Embryonic Losses in Beef and Dairy Cattle

José Luiz Moraes Vasconcelos 1, Fernando Henrique Sousa Aono2 & Marcos HenriqueColombo Pereira2

ABSTRACT

Background: The profitability of beef and dairy cattle is related to the herd reproductive efficiency, which can beaffected by reproductive disorders caused by infectious diseases. The immunization can be used to reduce reproductivelosses by the prevention of infectious diseases.

Review: Studies have shown that infectious diseases such as IBR, BVBV and Leptospirosis can cause pregnancylosses in beef and dairy cows and there are few information regarding the use of vaccination against IBR, BVDV andLeptospirosis in relation to the reproductive performance. The aim of this study was to evaluate the effect of thevaccine against IBR/BVDV/Leptospirosis (5.0 mL, i.m., Cattle Master® 4 + L5, Pfizer Animal Health, Lincoln, USA) atthe beginnig of the TAI protocol on pregnancy rate and pregnancy losses in beef and lactating dairy cows. Fiveexperiments were performed and all cows were inseminated at fixed time (TAI), in beef cows (n = 6145) the pregnancycheck was performed at 30 and 120 days post insemination (Exp. 01, 02, 03) and in dairy cows (n = 1960) at 30 and71 days post insemination (Exp. 04, 05). The experiment 01 was performed to evaluate the rate of pregnancy losses,without any vaccination schedule, and were evaluated 3464 pregnancies in 20 properties. In experiment 02 and 04were used cows never vaccinated, randomly assigned to receive or not the vaccine, following vaccination schedule:first dose at the beginning of the TAI protocol; second dose at the time of first diagnosis of gestation (30 days postTAI). In experiment 03 the pre – vaccination scheme was used within cows that already have a biannual vaccinationfor leptospirosis, and cows were randomly assigned to receive or not the vaccine against IBR / BVD / Leptospirosisthirty days before the start of the TAI protocol and the second dose at the beginning of the TAI protocol. In experiment05 cows that received vaccination against IBR / BVD / Leptospirosis in its annual health program, were randomlyassigned to receive a single dose of vaccine against IBR / BVD / Leptospirosis at the beginning of the TAI protocol.Data were analyzed by Logistic Regression in SAS. In experiment 01, were detected farm effect (P < 0.05) (resultsranging from 1.45% to 12.16%) and order effect (P < 0.05) (Primiparous: 9.03%; Multiparous: 5.06%) on the rate ofpregnancy loss between 30 and 120 days. In Experiment 02, treatment affected (P < 0.01) pregnancy rate at 30(Control: 53.2% and Vaccinated: 57.4%) and 120 days (Control: 48.3% and Vaccinated: 53.5%). In Experiment 03 wasdetected a tendency of effect (P < 0.10) of treatment on pregnancy rate at 30 (Control: 52.9%; Vaccinated: 59.7%) and120 days (Control: 50.0%; Vaccine: 57.7%). In experiment 04, animals that received the vaccine had a higher (P <0.05) pregnancy rate (44.4% vs. 37.6%) on day 30; higher (P < 0.01) pregnancy rate (41.1% vs. 31.7%) on day 71 andlower (P < 0.01) pregnancy loss (7.4% vs. 15.6%) between 30 and 71 days of gestation than the control group. Inexperiment 05, vaccination in the TAI protocol did not improve (P=0.8) the pregnancy rate on day 30 (34% vs. 34.7%);on day 71 (P=0.88) (30.6% vs. 30.1%) and in the pregnancy loss (P=0.19) (13.2% vs. 9.9%) compared with thecontrol group.

Conclusion: These data show that the use of Cattle Master® 4 + L5 had a positive impact on pregnancy rate, and incows that already received Cattle Master® 4 + L5 in their annual program, another vaccination in the beginning of theTAI protocol is not necessary.

Keywords: TAI, pregnancy rate, IBR, BVDV, Lepitospirosis, pregnancy loss.

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I. INTRODUCTION

II. MATERIALS AND METHODS

III. RESULTS

IV. CONCLUSIONS

I. INTRODUCTION

The profitability of production systems for beef or dairy cattle is related to the herd reproductive efficiency,which can be affected by reproductive disorders caused by infectious diseases.

Studies had show that approximately 40 – 50% of the causes of pregnancy loss are related to infectiousdiseases and among the infectious agents the IBR, BVDV and leptospirosis have been associates with reproductivedisorders [1].

The IBR virus has a negative effect on fertility, because its influence in the embryo quality, embryo deathand abortion [5,6]. It is estimated that 70 – 90% of BVDV infections occur without the manifestation of clinical signsand the major economic problem is due to the intrauterine infection, which may be associated with embryonic deathand abortion [3,4]. Leptospirosis can also cause fetal death, abortions and infertility [2]. The aim of this study was toevaluate the rate of pregnancy losses and the effect of the vaccination against reproductive diseases in the beginningof TAI protocol on the pregnancy rate in beef and lactating dairy cows.

II. MATERIALS AND METHODS

Were conduced 05 experiments to determine the effect of vaccination against IBR, BVDV, Leptospirosis,PI3, BRSV on pregnancy rate and pregnancy loss of beef cows (n=6145) and lactating dairy cows (n=1960) after TAI.

In experiment 01 and 02 were used cows never vaccinated against reproductive diseases and in experiment03 cows received immunization for Leptospirosis biannually. In experiments 01, 02 and 03 the cows received thesame TAI protocol: [D0- 2mg of estradiol benzoate (Estrogin®); and insertion of intravaginal progesterone devicecontaining 1.9g of progesterone (CIDR®); D7-dinoprost tromethamine (PGF2α, 12.5mg, Lutalyse®); D9- device withdrawal,injection of estradiol cypionate (1mg, ECP®) and calf removal; D11-TAI]. The evaluation of pregnancy was performedat 30 and 120 days post TAI. In experiment 01, any vaccination schedule was used and 3464 pregnancies in 20properties were evaluated to determine the rate of pregnancy losses between 30 and 120 days. The experiment 02,was performed in 07 properties, were used 2384 cows, randomly divided into the same group to receive or not thevaccine against IBR / BVD / Leptospirosis (5.0 mL, im, Cattle Master® 4 + L5, Pfizer Animal Health, Lincoln, USA)following the vaccination schedule: first dose at the beginning of the TAI protocol and the second dose (booster) at thetime of first diagnosis of gestation (30 days post TAI). The experiment 03, was performed in 01 properties, were used297 cows randomly distributed within the same group to receive or not the vaccine against IBR / BVD / Leptospirosis,the first dose thirty days before the start of the TAI protocol and the second dose at the beginning of the TAI protocolfeaturing a schedule of pre-vaccination.

In dairy cows in the experiment 04 were used 1140 cows with milk production of 21.3 ± 8.2 Kg/day in 37properties that had never use vaccine against IBR/BVD/Leptospirosis and in the experiment 05 were used 820 cowswith milk production of 24.5 ± 8.7 Kg/day in 16 properties that utilize vaccination against IBR/BVD/Leptospirosis intheir annual sanitary program. In both experiments all cows were timed artificially inseminated (TAI) with the protocol:D0-estradiol cipionate injection (2mg, ECPα) and insertion of intravaginal progesterone device containing 1.9g of P

4

(CIDRα); D7-prostaglandin injection (12.5mg, Lutalyseα); D9–device withdrawal, injection of estradiol cipionate (1mg,ECPα) and; D11-TAI. At the beginning of the TAI protocol cows were randomly divided into two groups to receive or notone dose of vaccine (5.0 mL, im, Cattle Master® 4 + L5). At 30 days after TAI, was performed the first pregnancydiagnosis and revaccination of cows of the group vaccinated in experiment 04. The second diagnosis of pregnancy

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was performed after 41 days to determine the pregnancy rate of day 71 and pregnancy loss between 30 and 71 daysof gestation. Data were analyzed by Logistic Regression in SAS.

III. RESULTS

In experiment 01, were detected farm effect (P < 0.05) [results ranging from 1.45% (2/138) to 12.16% (9/74)] and order effect (P < 0.05) [Primiparous: 9.03% (13/144); Multiparous: 5.06% (168/3320)] on the rate of pregnancyloss between 30 and 120 days. In Experiment 02, treatment affected (P < 0.01) pregnancy rate at 30 [Control: 53.2%(662/1244) and Vaccinated: 57.4% (654/1140)] and 120 days [Control: 48.3% (567/1174) and Vaccinated: 53.5% (578/1080)]. In Experiment 03 was detected a tendency of effect (P < 0.10) of treatment on pregnancy rate at 30 [Control:52.9% (81/153); Vaccinated: 59.7% (86/144)] and 120 days [Control: 50.0% (75/150); Vaccine: 57.7% (82/142)]. Inexperiment 04, animals that received Cattle Master® 4 + L5 had a higher (P < 0.05) pregnancy rate [44.4% (257/579)vs. 37.6% (211/561)] on day 30; higher (P < 0.01) pregnancy rate [41.1% (238/579) vs. 31.7% (178/561)] on day 71and lower (P < 0.01) pregnancy loss [7.4% (19/257) vs. 15.6% (33/211)] between 30 and 71 days of gestation than thecontrol group. In experiment 05, vaccination in the TAI protocol did not improve (P = 0.8) the pregnancy rate on day 30[34% (131/385) vs. 34.7% (151/435)]; on day 71 (P = 0.88) [30.6% (118/385) vs. 30.1% (131/435)] and in the pregnancyloss (P = 0.19) [13.2% (20/435) vs. 9.9% (13/385)] compared with the control group.

IV. CONCLUSIONS

These results show that there are farms with different rates of pregnancy loss and the vaccination withCattle Master ® 4 + L5 had a positive impact on pregnancy rate. In cows that already received Cattle Master® 4 + L5in their annual sanitary program, another vaccination in the beginning of the TAI protocol is not necessary.

REFERENCES

1 Grooms D.L. 2010. Programas para controle de doenças infecciosas e melhoria do desempenho reprodutivo. In anais do XIV CursoNovos Enfoques na Produção e Reprodução de Bovinos (Uberlandia – Brasil).

2 Grooms D.L. 2010. Diagnóstico e controle de perdas reprodutivas causadas por leptospira spp. In anais do XIV Curso NovosEnfoques na Produção e Reprodução de Bovinos (Uberlandia – Brasil).

3 Grooms D.L., Bolin S.R., Coe P.H., Borges R.J. & Coutu C.E. 2007. Fetal protection against exposure to bovine viral diarrhea virusfollowing administration of a vaccine containing an inactivated bovine viral diarrhea virus fraction to cattle. American Journal of VeterinaryResearch. 68(12):1417-1422.

4 Grooms D.L. 2004. Reproductive consequences of infection with bovine viral diarrhea virus. The Veterinary Clinics of North America.Food Animal Practice. 20(1):5-19.

5 Kelling C.L. 2007. Viral Diseases of the Fetus. University of Nebraska – Lincoln. Published, as Chapter 50, in Current Therapy in LargeAnimal Theriogenology (2nd edition), edited by Robert S. Youngquist and Walter R. Threfall (St. Louis, MO: Saunders-Elsevier). 399–408.

6 Miller J.M. & Maaten Van Der M.J. 1986. Experimentally induced infectious bovine rhinotracheitis virus infection during early pregnancy:effect on the bovine corpus luteum and conceptus. American Journal of Veterinary Research. 47(2): 242-240.


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Heat Stress and Embryo Production in High-Producing Dairy Cows

Roberta Machado Ferreira1, Henderson Ayres1, Marcos Roberto Chiaratti2, Carlos AlbertoRodrigues3, Bruno Gonzalez Freitas1, Flávio Vieira Meirelles2 & Pietro Sampaio Baruselli1

ABSTRACT

Background: Heat stress (HS) can compromise the female reproductive system, resulting in a massive reduction ofreproductive performance. During the period of follicular growth, HS may compromise the oocyte, either because of a directeffect of elevated temperature on the gamete or because of changes in follicular function that would damage oocyte quality.Oocytes harvested from Holstein cows during summer have reduced ability to develop to the blastocyst stage than thoseharvested during winter. Nevertheless, although there are reports showing a clear effect of HS on oocyte competence todevelopment after IVF, the exact mechanisms by which the oocyte is compromised remain unknown.

Review: Resistance to HS is dependent on cow’s genotype. Bos indicus breeds (i.e. Gir) are more resistant to elevatedtemperature and humidity than breeds (i.e. Holstein) that evolved in a temperate climate. This resistant is, among otherfactors, due to the superior ability of certain breeds to regulate their body temperature. Thus, breed selection is present asa possible tool to minimize the effects of temperature and humidity on milk production and reproduction. On the other hand,Holstein cows are the most common breed used, even intropical countries (i.e. Brazil), because of their yield in milkproduction. As a result, there is a considerable effect upon milk production and reproductive performance through the yearin tropical and subtropical countries. A possible way to minimize this effect is the employment of reproductive biotechnologiessuch as fixed-time artificial insemination (FTAI) and embryo transfer (ET). FTAI has been shown to eliminate problems ofestrus detection caused by HS. Nevertheless, it is not enough to complete restore herd pregnancy rates because oocytesand early embryos have already been damaged by HS. In contrast, embryos three days after conception or older are lesssensitive to HS. Thus, ET has been used to improve cow’s reproductive performance during the hot months of the year (i.e.spring and summer). This is carried out by producing embryos during the fresh months but using them for ET during the HSperiod. Regarding fertility problems, it is recognized that repeat-breeder (RB) cows cause huge economic problems to thefarmer. These cows are characterized by poor fertilization rates and/or early embryonic loss. It is still not certainly knownwhy these cows become RB, when it is the critical period of the year for them, and what should be done to avoid thisproblem. We have observed that the conception rates in RB Holstein cows were greater after ET (41.7%) than after AI(17.9%). This indicates that ET may be an effective alternative to achieve satisfactory conception rates throughout the yearin RB cows, especially during periods of HS. Moreover, when three categories of Holstein cows (heifers, high-producingcows in peak lactation and RB) were subjected to ovum pick up during the summer and the winter, blastocyst rates andquality were significantly affected by both, category and period, being RB cows mostly affected during the summer.Moreover, lower amounts of mitochondrial DNA were found in RB oocytes, suggesting that the low reproductive performanceobserved in these cows is related to some injure in oocyte quality.

Conclusion: HS negatively affects the physiology and fertility of dairy cows and heifers. Concerning about thereproductive field, the effects of HS were detected in vitro and in vivo on oocytes and embryos and also on conceptionrates. Moreover, RB Holstein cows seem to be even more sensitive to HS and this can probably be related to theiroocyte potential into develop in blastocysts. Therefore, efforts should be taken in order to improve the environmentalconditions and heat resistance of dairy cows in tropical and subtropical areas. Studies are been conducted to understandthe molecular level by which HS disrupt reproduction aiming to develop methods to attenuate or reverse HSconsequences upon fertility of dairy cows.

Keywords: bovine, heat-stress, Holstein, oocyte quality, repeat-breeder, in vitro embryo production.

1Laboratório de Reprodução Animal, VRA, FMVZ-USP, São Paulo-SP, Brazil, 2Laboratório de Morfofisiologia Molecular e Desenvolvimento,ZAB, FZEA-USP, Pirassununga-SP, Brazil, 3SAMVET, São Carlos-SP, Brazil. Address. Av. Prof. Dr. Orlando Marques de Paiva, 87,Butantã, São Paulo-SP, Zip Code 05508-000. CORRESPONDENCE: R. M. Ferreira: [E-mail: [email protected];[email protected]].

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I. INTRODUCTION

II. HEAT STRESS RESISTANCE AND COW GENOTYPE

III. HEAT STRESS IN BOS TAURUS CATTLE

IV. EFFECT OF MATERNAL HEAT-STRESS ON DIFFERENT CATEGORIES OF BOSTAURUS CATTLE

V. CONCLUSION

I. INTRODUCTION

Heat stress (HS) can be defined as the sum of forces exterior to a homeothermic animal that acts todislocate body temperature from the resting state [37]. The physiological adaptations that homeotherms undergoduring HS, can compromise several physiological important systems, including reproduction. In bovine, hyperthermiacan affect cellular function in various tissues of the female reproductive tract [18,36]. Moreover, redistribution of bloodflow from the viscera to the periphery during HS can disrupt ovarian and uterine physiology, affecting reproductiveperformance [4,8,19].

Focusing on reproduction, studies have shown that HS during the period of follicular growth has the potentialto compromise the oocyte, either because of a direct effect of elevated temperature on the oocyte or because ofchanges in follicular function that would compromise oocyte quality (reviewed by [19]). Oocytes harvested fromfollicles of Holstein (Bos taurus) cows during summer had reduced ability to develop to the blastocyst stage after invitro fertilization (IVF) than oocytes harvested during winter [27,3]. Nevertheless, although there are reports showinga clear effect of HS on oocyte competence to development after IVF, the exact mechanisms by which the oocyte iscompromised remain unknown.

Previous studies have shown that the exposure of bovine cumulus–oocyte complexes (COCs) to elevatedtemperature during the first 12 h of in vitro maturation (IVM) disrupts cytoskeleton architecture, reduces oocytenuclear maturation [33], and induces oocyte death through apoptosis [31,32]. These deleterious effects of HS decreasedthe rate of embryo development into blastocyst following IVF [31,10]. Moreover, a detrimental effect of HS on blastocystquality or cell number is consistent with reports showing reduced in vitro or in vivo embryonic development during HS[2,3,18,26].

II. HEAT STRESS RESISTANCE AND COW GENOTYPE

It is known that the resistance to HS is mainly determined by the cow’s genotype. B. indicus cattle breedshave been shown to be more resistant to tropical conditions (i.e. elevated temperature and humidity) than breeds thatevolved in a temperate climate (i.e. B. Taurus, as Holstein). Essentially, the adaptation of certain breed to heatconditions is due to the superior ability of thermotolerant breeds to regulate their body temperature [1,5,17,14].However, it has been observed that even thermotolerant breeds such as Nelore (B. indicus) cows have a deleteriouseffect on oocyte quality, assessed by the in vitro embryo production rate, when their oocyte are collected during theend of summer [11].

To further clarify the above finding, Torres-Júnior et al. [35] designed an experiment with B. indicus dairycows (Gir breed) aiming to determine immediate and delayed effects of HS on follicular dynamics, oocyte competence,and hormonal profiles. In this study, Gir cows were first subjected to an adaptive thermoneutral period of 28 days intie-stalls (Phase I) to be then allocated into two treatments (Phase II). The Control group (n = 5) was maintained inshaded tie-stalls (ambient temperature), while the HS cows were placed in an environmental chamber at 38.8°C and80% relative humidity (RH) during the day and 30.8°C, 80% RH during the night for 28 days. During Phase III, cowsfrom both groups were allocated in tie-stalls (Days 28–42) followed by pasture (Days 42–147) under thermoneutrality(Figure 1).

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Figure 1. Environmental dry bulb temperature (solid lines) and rectal temperature (dotted lines) in thermoneutral control (CG) or heat-stress(HS) treatments. Means ± S.E.M. (Adapted from Torres-Júnior et al., 2008).

In each phase, weekly ovum pick up (OPU) sessions were carried out to evaluate follicular development,COCs morphology, and developmental competence by IVP. In addition, serum concentrations of progesterone (P4)and cortisol were assessed. Exposure of Gir cows to HS had no immediate effect on reproductive function, but adelayed deleterious effect on ovarian follicular growth, hormone concentrations, and oocyte competence was reported.As shown in Figure 2, HS resulted in an increase of the first and second largest follicles diameter from Days 28 to 49and reduced P4 concentration up to Day 28, as compared to thermoneutral Control group.

Cows exposed to HS had longer periods of non-cyclic activity (P4 < 1 ng/mL), as well as shorter estrouscycles. Indeed, HS induced follicular codominance (increased the number of follicles larger than nine millimeters) andreduced the estrous cycle length. However, HS did not affect cortisol concentration as compared to Control group.Although HS had no significant effect on cleavage rate, it reduced blastocyst development during Phase III of theexperiment. In conclusion, long-term exposure of Gir (B. indicus) cows to HS had a delayed deleterious effect onovarian follicular dynamics and oocyte competence to develop into blastocyst.

A previous seasonal experiment have demonstrated that once the pool of ovarian oocytes is damaged bysummer HS, two or three estrous cycles are required (after the end of HS) to restore the follicular pool and oocytequality [30]. However, the study described above [35] shows the existence of a carry-over effect of HS on blastocystproduction maintained up to 105 days after the end of HS. Therefore, it seems that follicles and oocytes are damagedby HS during early stages of folliculogenesis, with a delayed deleterious effect on ovarian function.

III. HEAT STRESS IN BOS TAURUS CATTLE

In Brazil, farmers have been constantly investing on breed selection aiming to improve the potential of milkproduction in dairy cows. However, some important characteristics related to reproduction and heat resistance havebeen left behind. Cattle genetically adapted to tropical and subtropical areas (as the Gir cattle) have been graduallyreplaced by high-producing, unadapted breeds, such as Holstein (B. taurus) cows. It is known, for instance, that thedegree by which HS reduces endometrial [23], lymphocyte [20,25] and embryonic function [25,34] is greater for B.taurus than B. indicus cattle. Since breed differences in thermal resistance extend to the cellular level, such replacement

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can severely impact herd’s susceptibility to HS. This scenario can be even more serious if we consider that HSeffects tend to increase in magnitude with the continued global warming that is already occurring.

Several studies have shown that the use of fixed-time artificial insemination (FTAI) protocols can eliminateproblems of estrus detection caused by HS but is not sufficient to restore herd pregnancy rates to a level seen infresh weather. As previously described, oocytes and embryos at early stages of development are extremely sensitiveto HS and this can be more pronounced in unadapted breeds. Embryos three days after conception or older are lesssensitive to HS [9,19]. A potential tool that has been used to improve summer fertility by bypassing the effects of HSon early embryonic development is embryo transfer (ET). Thus, aiming to create a panorama on how HS is affectinghigh-producing Holstein cows reproduction in Brazilian environmental conditions, we performed a retrospective studyto assess conception rates after artificial insemination (AI) and ET in different seasons (months) of the year [28]. Thestudy was carried out in a commercial farm (Agrindus S/A, Descalvado, Brazil) located in south-east Brazil. The dataset comprised 12,875 AIs and 4,822 ETs, performed from 2001 to 2006. We observed that when AI is employed, theconception rates decrease during the months of HS (Figure 3). However, the use of ET increases the overall conceptionrates and is efficient to keep these rates more homogenous over the four different season of the year (Figures 3 and4).

Figure 2. Diameter of the dominant follicle (DF) and the largest subordinate follicle (SF) and serum progesterone (P4) concentration (grayarea) of Gir (Bos indicus) cows exposed to thermoneutral (CG; bottom panel) or heat-stress (HS; top panel) treatments. (Adapted fromTorres-Júnior et al., 2008).

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Figure 3. Conception rates of high-producing Holstein cows per month following artificial insemination (AI) or embryo transfer (ET), duringa six-year period. a ‘“ b differ within month (P<0.05; Adapted from Rodrigues et al., 2007a).

Figure 4. General conception rates of high-producing Holstein cows following artificial insemination or embryo transfer, during a six-yearperiod a ‘“ b differ within month (P<0.05; Adapted from Rodrigues et al., 2007a).

In another retrospective study we evaluated the embryonic loss between 30 and 60 days of pregnancy, inhigh-producing Holstein cows subjected to AI or ET during summer and winter [13]. The study was carried out in thesame farm and the data set comprised 5,040 AIs and 2,109 ETs performed from 2004 to 2008. Regardless thebiotechnic (AI or ET), pregnancy loss was greater during the spring-summer than during the fall-winter [20.3% (593/2916) vs 16.8% (709/4234); P = 0.002]. However, cows submitted to ET had greater (P = 0.001) pregnancy loss(20.5%; 432/2109) than cows artificially inseminated (17.3%; 870/5040; [13]). When conception rates from samecows and seasons were evaluated we observed a decrease of this rate during spring-summer using either ET or AI.

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Nevertheless, the conception rate following ET was always greater than after AI (Figure 5, unpublished data). Thus,in spite of greater embryo loss compared to AI, the use of ET enabled an improvement on cow’s reproductiveperformance during the hot months of the year (i.e. spring and summer). Therefore, an interesting strategy to keepsimilar pregnancy outcomes along the year would be to produce embryos during the fresh months and use them forET during the HS period.

Regarding fertility problems, it is recognized that repeat-breeder (RB) cows cause huge economic problemsto the farmer [21]. Repeat-breeder cows are commonly referred to as sub-fertile animals without any anatomic orinfectious abnormality that do not become pregnant by the third breeding. These cows are characterized by poorfertilization rates [15,24] and/or early embryonic loss [22,16]. It is still not certainly known why these cows becomeRB, when it is the critical period of the year for them, and what should be done to avoid this problem. Thus, weperformed a second retrospective study, similar that described above and using the same data, to evaluate only thepattern of RB conception rates following AI or ET through the year ([29]; Figure 6). We observed that conception ratesin RB Holstein cows were greater after ET (41.7%; 1609/3858) than after AI (17.9%; 1019/5693). Also, pregnancy losswas similar among RB and non-RB cows submitted to AI or ET [13]. This indicates that ET may be an effectivealternative to achieve satisfactory conception rates throughout the year in RB cows, especially during periods of HS.

IV. EFFECT OF MATERNAL HEAT-STRESS ON DIFFERENT CATEGORIES OF BOSTAURUS CATTLE

Recently, we performed an experiment during the fresh and hot periods of the year aiming to assess theoutcomes at OPU and IVP among different categories of Holstein cattle [heifers (H), high-producing cows in peaklactation (PL) and RB] subjected or not to summer HS. It was hypothesized that i) Holstein cattle under HS wouldhave compromised IVP compared to cattle in fresh conditions; ii) heifers would have greater IVP than PL and RBcows; iii) RB would have compromised IVP compared to non-RB cows. The follicular wave emergence of all animals

Figure 5. Conception rate and pregnancy loss of high-producing Holstein cows per period (spring-summer and fall-winter) following artificialinsemination (AI) or embryo transfer (ET), during a five-year period (Adapted from Freitas et al., 2010; in press).

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was synchronized and OPU was performed by a single technician. Respiration rate, rectal and cutaneous temperaturewere recorded when cattle started synchronization (five days before OPU). Part of the retrieved oocytes went throughIVP and the remainder was kept at -80oC without cumulus cells. Semen from a single batch of a Holstein bullpreviously tested was used in IVP. On the eighth day of embryo culture, zona pellucida-intact embryos were assayedto quantify the total cell number and to assess nuclear fragmentation rate by TUNEL.

Heifers and cows had similar respiration rate during the fresh period, but cows had their respiration rateincreased during HS (Table 1; [12]). Regarding rectal temperature, heifers kept it similar in summer and winter;however, PL and RB cows had their temperature increased during HS (Table 2; [12]). Heifers and cows had similarnumber of oocytes and viable oocytes recovered by OPU during the winter. On the other hand, during HS thesenumbers were reduced only in cows. At IVP, the cleavage rate was similar among categories (H = 51%, PL = 37.5%,RB = 41.6%; P = 0.18) and periods (winter = 42.3%, summer = 45.8; P = 0.45). However, the blastocyst rate wasaffected by both, category and period (Table 3; [12]). Hatching blastocyst rate and blastocyst cell number (Day 8) weregreater during the winter (23%, 60/261 and 231 ± 7) than summer (6.5%, 7/107; P = 0.03 and 253 ± 12; P < 0.01). Also,blastocyst cell number was greater in heifers (253 ± 12) than PL (203 ± 10) and RB cows (207 ± 8; P < 0.01). Theblastocyst nuclear fragmentation rate (Table 4; [12]) was greater and the number of copies of mitochondrial DNA(mtDNA) was reduced for RB cows during the summer (Figure 7; [12]), evidencing an oocyte quality disruption [6,7].

Figure 6. Conception rate of high-producing Holstein repeat-breeder cows per month following artificial insemination (AI) or embryo transfer(ET), during a six-year period (Adapted from Rodrigues et al., 2007b).

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Figure 7. Oocyte mitochondrial DNA (mtDNA) content of Holstein cattle categories during winter and summer (relative to average heiferswithin winter). Circles on the graph depict mean; crosses depict minimum and maximal values; thick bars depict median, whereas squaresdepict 25 and 75 percentiles (upper and lower thick bars) and whiskers depict 5 and 95 percentiles. a ‘“ b differ within season (P<0.01) and* differ within category (P=0.0004). (Adapted from Ferreira et al., 2010; in press).

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Table 1. Respiration rate (breaths/minute) of Holstein cattle of different categories (heifers, high-producingcows in peak lactation and repeat-breeder cows) during winter and summer 2009. (Adapted from Ferreira et al.,2010; in press).

Heifer Peak lactation Repeat Breeder

Winter 40.59 ± 1.04 43.74 ± 1.49b 43.10 ± 1.23b

(n = 34) (n = 32) (n = 31)

Summer 44.78 ± 1.30B 72.86 ± 2.25aA 73.83 ± 1.41aA

(n = 36) (n = 37) (n = 36)

Mean (±S.E.) values within rows with different superscript letters (A = B) differ significantly (P<0.0001)

Mean (±S.E.) values within columns with different superscript letters (a = b) differ significantly (P<0.0001)

Category*season: P<0.0001

Table 2. Rectal temperature (°C) of Holstein cattle of different categories (heifers, high-producing cows in peaklactation and repeat-breeder cows) during winter and summer 2009. (Adapted from Ferreira et al., 2010; inpress).


Winter 38.65 ± 0.07B 39.21 ± 0.07bA 38.81 ± 0.07bB

(n = 34) (n = 32) (n = 31)

Summer 38.61 ± 0.06B 39.76 ± 0.12aA 39.51 ± 0.11aA

(n = 36) (n = 37) (n = 36)




Table 3. Blastocyst rate of Holstein cattle of different categories (heifers, high-producing cows in peak lactationand repeat-breeder cows) during winter and summer 2009. (Adapted from Ferreira et al., 2010; in press).


Winter 30.3%aA 22.0%aB 22.5%aB

(74/244) (42/191) (93/413)

Summer 23.3%bA 14.6%bB 7.9%bC

(35/150) (15/103) (14/177)




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Table 4. Nuclear fragmentation rate in blastocysts of Holstein cattle of different categories (heifers, high-producing cows in peak lactation and repeat-breeder cows) during winter and summer 2009. (Adapted fromFerreira et al., 2010; in press).


Winter 1.82% 2.48% 2.15%b

(n = 61) (n = 49) (n = 78)

Summer 2.51%B 2.68%B 3.85%Aa

(n = 13) (n = 14) (n = 14)




V. CONCLUSION

In conclusion, HS negatively affects the physiology and fertility of dairy cows and heifers. Concerningabout the reproductive field, the effects of HS were detected in vitro and in vivo on oocytes and embryos, and also onconception rates. Moreover, RB Holstein cows seem to be even more sensitive to HS. Therefore, efforts should betaken in order to improve the environmental conditions and heat resistance of dairy cows in tropical and subtropicalareas. Also, studies are been conducted to understand the molecular level by which HS disrupt reproduction and todevelop methods to attenuate or reverse HS consequences upon fertility.

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