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Fertility-associated antigen on Nelore bull sperm and reproductive outcomes following first-service fixed-time AI of Nelore cows and heifers
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Theriogenology 77 (2012) 389 –394 www.theriojournal.com
Fertility-associated antigen on Nelore bull sperm and reproductive outcomes following first-service fixed-time AI of Nelore cows and heifers J.C. Daltona,*, L. Deragonb, J.L.M. Vasconcelosc, C.N. Lopesd, R.F.G. Perese, A. Ahmadzadehf a
Department of Animal and Veterinary Science, University of Idaho, Caldwell, Idaho, USA b Alta Genetics, Uberaba, Minas Gerais, Brazil c Departamento de Produçáo Animal, Faculdade de Medicina Veterinária e Zootecnia-UNESP, Botucatu, São Paulo, Brazil d Fazenda Anita, Mato Grosso do Sul, Brazil e Agropecuária Fazenda Brazil, Barra do Garças, Mato Grosso, Brazil f Department of Animal and Veterinary Science, University of Idaho, Moscow, Idaho, USA Received 27 May 2011; received in revised form 29 July 2011; accepted 4 August 2011
Abstract The objective was to determine whether the presence of fertility-associated antigen (FAA) on sperm collected from Nelore (Bos indicus) bulls can be used to assess potential fertility of sperm for use at first-service fixed-time AI (TAI). Six Nelore bulls were selected based on FAA status (FAA-negative: N ⫽ 3; FAA-positive: N ⫽ 3) and the ability to produce neat semen with ⱖ 70% morphologically normal sperm and 60% estimated progressive motility before cryopreservation. In Experiment 1, suckled multiparous Nelore cows (N ⫽ 835) were evaluated for body condition score (BCS) and received an intravaginal progesterone device (CIDR) and 2.0 mg of estradiol benzoate (Day 0). On Day 9 the CIDR was removed, 12.5 mg of PGF2␣ and 0.5 mg of estradiol cypionate were administered, and calves were removed for 48 h. All cows received TAI on Day 11 (48 h after CIDR removal). Pregnancy per TAI (P/TAI) was not different between FAA-positive and FAA-negative bulls (41.5% vs. 39.3%, respectively). There was an effect of AI technician on P/TAI (36.0% vs. 43.9%; P ⬍ 0.05) and BCS tended to affect P/TAI (P ⫽ 0.09), as cows with BCS ⱖ 2.75 were 1.4 times more likely to become pregnant compared with cows with BCS ⬍ 2.75. In Experiment 2, nulliparous Nelore heifers (N ⫽ 617) were evaluated for BCS and received a CIDR and estradiol benzoate (2.0 mg) on Day 0. On Day 7, all heifers received PGF2␣ (12.5 mg). On Day 9, CIDR inserts were removed and all heifers received estradiol cypionate (0.6 mg) and 200 IU eCG. All heifers received TAI on Day 11 (48 h after CIDR removal). Pregnancy/TAI was different (P ⫽ 0.04) between FAA-positive and FAA-negative bulls (33.7% vs. 40.7%, respectively). Presence of FAA on sperm was unsuccessful in assessing the potential fertility of sperm for use in TAI. © 2012 Elsevier Inc. All rights reserved. Keywords: Sperm; Fertility-associated antigen; Fixed-time AI; Bos indicus; Cattle
1. Introduction
* Corresponding author. Tel.: ⫹1 208 459 6365; fax: ⫹1 208 454 7612. E-mail address: [email protected] (J.C. Dalton). 0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.08.011
Synchronization programs that include fixed-time artificial insemination (TAI) provide an organized and efficient approach to administering AI. Consequently, beef and dairy cattle producers throughout the world are interested in identifying the most fertile bulls for
390
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TAI. Although many sperm attributes necessary for fertilization are known, including normal morphology, progressive motility, capacity for hyperactive motility, acrosomal enzymes, and chromatin integrity, many other attributes remain unknown [1]. Heparin-binding proteins (HBP) are secreted by the seminal vesicles, prostate, and Cowper’s glands, and coat sperm during ejaculation [2,3]. In 1994, Bellin and coworkers [4] reported that the distribution of specific forms of HBP on sperm corresponded to fertility potential of bulls used for natural service. Different patterns of HBP on bovine sperm have since been described [5,6]. Bulls with detectable fertility-associated antigen (FAA), a 31kDa molecular weight protein on sperm, were 9 to 40 percentage points more fertile (following natural service) than bulls producing sperm lacking FAA [5,6]. Sprott et al. [7] used 25 bulls of mixed breeds, including Bos taurus, Bos taurus ⫻ Bos indicus, and Bos indicus (Brahman) to investigate whether FAA could be used to assess the potential fertility of sperm to be used for AI. Bulls with sperm that were FAApositive were 7 to 9 percentage points more fertile following first-service AI than bulls producing sperm lacking FAA [7]. Collectively, the results of Bellin et al. [5,6] and Sprott et al. [7] provided evidence that FAA appeared to be an important trait of sperm that may be used in the evaluation of fertility potential. Nevertheless, the use of sophisticated techniques to determine the presence of FAA [6,7] and other proteins [8 –11] associated with fertility is not practical outside the research laboratory. McCauley et al. [12], however, reported on the development and validation of a lateral flow cassette for the rapid determination of FAA status in bulls, without the requirement for time-consuming, intensive techniques that must be performed in a research laboratory. Taken together with the realization that identification of novel spermatozoal or seminal attributes associated with male fertility may increase the accuracy of fertility estimation [13], the objective of this research was to use the commercially available lateral flow cassette to help determine whether the presence of FAA on sperm collected from Nelore (Bos indicus) bulls can be used to assess potential fertility of sperm for use at first-service TAI in multiparous Nelore cows and nulliparous Nelore heifers. 2. Materials and methods 2.1. Bulls, semen collection, and determination of spermatozoal FAA status Ejaculates from Nelore bulls (N ⫽ 49; range in age: 2 yr 8 mo to 11 yr) housed at Alta Genetics, Uberaba,
MG, Brazil, were collected by artificial vagina. Each ejaculate was preceded by two false mounts separated by 2 min of restraint. Immediately after collection and determination of ejaculate volume, 2.0 mL of neat semen was removed for use in a lateral flow cassette (ReproTest, Midland Bioproducts Corporation, Boone, IA, USA) which facilitated rapid on-site determination of spermatozoal FAA status as described previously [12]. Six Nelore bulls were selected for the field trial, based on presence or absence of FAA (FAA-positive: N ⫽ 3; FAA-negative: N ⫽ 3, respectively), on the ability to produce neat semen with ⱖ 70% morphologically normal sperm, and 60% estimated progressive motility before cryopreservation. 2.2. Semen evaluation, extension, and cryopreservation Immediately after collection, the vial containing the ejaculate was placed in a water bath at 32 °C. Concentration was determined using a spectrophotometer calibrated for bovine sperm using a hemacytometer. Motility was estimated to the nearest 5% after viewing several microscopic fields at magnification 250 X using a phase contrast microscope equipped with a heated stage (37 °C). The neat semen and extender (AndroMed, Minitube, Tiefenbach, Germany) were maintained in the same water bath. The semen was diluted 1:1 with extender and remained in the water bath for not more than 10 min. Subsequently, the semen was fully extended to 120 ⫻ 106 sperm per mL, and passively cooled to 18 °C for approximately 1 h. To facilitate sperm morphologic analysis, wet smears of extended semen fixed with glutaraldehyde were prepared [14]. Sperm abnormalities were quantified using differential interference contrast microscopy at magnification 1200 X and oil immersion. Differential counts of 100 cells from each of two wet smears of the fixed sample were averaged. Specific cell abnormalities were classified as described by Blom [15,16]. The extended semen was packaged in 0.25-mL straws (30 ⫻ 106 sperm), and the straws were placed in a cooling cabinet at 5 °C for 3.5 h for equilibration. Semen was cryopreserved using a programmable freezer (DigiCool, Instruments de Medecine Veterinaire, l’Aigle, France) and was stored in liquid nitrogen (⫺196 °C) until use. For evaluation of progressive motility postthaw, sample straws were thawed in water at 37 °C for 1 min. Sperm motility was estimated immediately after thawing and after 3 h of incubation at 37 °C as previously described.
J.C. Dalton et al. / Theriogenology 77 (2012) 389 –394
2.3. Experiment 1: animals and fixed-time AI protocol Suckled multiparous Nelore cows (N ⫽ 835) 40 to 60 days postpartum, were evaluated for body condition score (BCS; one to five scale; [17]) and enrolled in a first-service TAI program. All cows were maintained on Brachiaria decumbens pasture at a commercial beef cattle ranch in Mato Grosso do Sul, Brazil, with ad libitum access to water and mineral during the experimental period. On Day 0, the synchronization protocol began with a new intravaginal insert containing 1.9 g progesterone (CIDR; Pfizer Animal Health, São Paulo, SP, Brazil; new CIDR: first use), a CIDR that had been used previously in a 9-day synchronization protocol (second use), or a CIDR that had been used in two previous 9-day synchronization protocols (third use), and estradiol benzoate (2.0 mg im; Estrogin, Farmavet, São Paulo, SP, Brazil). On Day 9, CIDR inserts were removed and all cows were treated with PGF2␣ (PGF; 12.5 mg im; Lutalyse, Pfizer Animal Health, São Paulo, SP, Brazil) and estradiol cypionate (0.5 mg im; ECP, Pfizer Animal Health). Immediately after CIDR withdrawal, calves were removed from cows for 48 h. During the period of calf removal, calves were held in pens out of sight from their dams, and had ad libitum access to water. On Day 11 (48 h after CIDR removal), all cows received TAI. Two experienced technicians performed the inseminations, with each technician using semen from each bull. To randomize semen, AI technicians alternated usage of one straw from each bull. Calves were reunited with their dams immediately after TAI. Pregnancy was diagnosed by transrectal ultrasonography 28 to 35 days after TAI. 2.4. Experiment 2: animals and fixed-time AI protocol Nulliparous Nelore heifers (N ⫽ 617; 24 to 28 mo of age; 300 to 340 kg body weight) were evaluated for BCS (one to five scale; [17]) and enrolled in a firstservice TAI program. All heifers were maintained on Brachiaria decumbens pasture at a commercial beef cattle ranch in Mato Grosso, Brazil, with ad libitum access to water and mineral during the experimental period. On Day 0, heifers began the synchronization protocol with a second-use (9-day previously used) intravaginal progesterone insert (CIDR; Pfizer Animal Health) and estradiol benzoate (2.0 mg im; Estrogin, Farmavet). On Day 7, all heifers were treated with PGF2␣ (12.5 mg im; Lutalyse, Pfizer Animal Health). On Day 9, CIDR inserts were removed and all heifers were treated with ECP (0.6 mg im; Pfizer Animal
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Health) and equine chorionic gonadotropin (200 IU im; Folligon; Intervet Schering-Plough, São Paulo, SP, Brazil). On Day 11, all heifers received TAI 48 h after CIDR removal. Two experienced technicians performed the inseminations, with each technician using semen from each bull. To randomize semen, AI technicians alternated usage of one straw from each bull. Pregnancy was diagnosed by transrectal ultrasonography 28 to 35 days after TAI. 2.5. Statistical analysis For both experiments, pregnancy per TAI (P/TAI) data were analyzed by logistic regression using SAS [18]. For multiparous cows (Experiment 1), the full statistical model included the effects of treatment (FAA-positive or FAA-negative), sire (six bulls), technician (two inseminators), BCS (⬍ 2.75 and ⱖ 2.75, on scale of one to five), CIDR (new or used), treatment by technician, treatment by BCS, and technician by BCS. All two-way interactions with a P value ⬎ 0.25 were eliminated from the model; hence independent variables included in the final model were treatment, sire, technician, BCS, and CIDR. For nulliparous heifers (Experiment 2), the full statistical model included the effects of treatment (FAA-positive or FAA-negative), sire (six bulls), technician (two inseminators), BCS (⬍ 3.0 and ⱖ 3.0, on scale of one to five), treatment by technician, treatment by BCS, and technician by BCS. All two-way interaction variables with a P value ⬎ 0.25 were eliminated from the model; hence independent variables included in the final model were treatment, sire, technician, and BCS. 3. Results and discussion 3.1. FAA status of Nelore bulls This was the first trial conducted using the commercially available lateral flow cassette for determination of FAA status and comparison of fertility following first-service TAI in multiparous and nulliparous Bos indicus cattle using cryopreserved semen. Fifty tests were conducted on ejaculates from 49 bulls. Fortyseven tests (on ejaculates from 47 bulls) provided a distinct determination of FAA status. Three tests on ejaculates from two bulls were inconclusive (due to failure of the test, as evidenced by lack of a visible control band on the cassette). Five bulls (11%; 5/47) were FAA-negative, whereas 42 bulls (89%; 42/47) were FAA-positive. The proportion of FAA-negative Nelore bulls (11%) was similar to a previous report
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(12% FAA-negative; [6]), but less than in other studies using Bos taurus, Bos taurus ⫻ Bos indicus, and Brahman (Bos indicus) bulls (28% FAA-negative, [7]; 22% FAA-negative, [19]). Caution must be used when interpreting FAA status of a group, however, as the range of FAA-negative bulls [6] was 0 to 50% (depending on the group of bulls tested). Furthermore, it is possible that the apparent differences between studies in average proportion of FAA-negative bulls may be related to intensity of selection for overall fertility, as 100% of Holstein bulls tested and housed at AI centers were FAA-positive [6], compared with 89% of Nelore bulls tested and housed at the AI center in the present study. In contrast, the bulls used by Sprott et al. [7,19] were not housed at an AI center; consequently, it is possible that less intense selection for fertility resulted in a greater proportion of FAA-negative bulls [7,19]. 3.2. Experiment 1: fertility following TAI of suckled multiparous Nelore cows Fertility, as measured by P/TAI, was not different between FAA-positive and FAA-negative bulls (41.5% vs. 39.3%, respectively). These results were not consistent with the beneficial effect of FAA-positive bulls on pregnancy rates (pooled) to first AI in beef cows [7]; however, these results agreed with the observation that some FAA-positive bulls yield pregnancy rates similar to those of FAA-negative bulls following AI [7]. Within the bulls used in the present study, FAA-negative status was not a limiting factor of fertility. Apparently, sperm from both FAA-positive and FAA-negative bulls possessed sufficient levels of known and unknown characteristics [1] necessary to achieve similar fertility. Given that the overall objective of an AI center quality control program is to minimize variation in sire fertility [20], that similar P/TAI resulted following the use of sperm (with and without detectable FAA) from bulls housed at an AI center was not entirely unexpected. There was no effect of type of CIDR (new: first-use, 9-days or 18-days previously used: second-use or thirduse, respectively) on P/TAI, which agrees with previous reports [21,22] investigating previously used CIDR inserts in synchronization protocols with Bos indicus cattle. There was an effect of AI technician on P/TAI (36.0% vs. 43.9%; N ⫽ 375 and N ⫽ 460 respectively; P ⬍ 0.05); however, there was no AI technician by FAA status (treatment) interaction. Previous reports of differences in AI technician success have been reported [23–25]. Cows entered the breeding chute in random order and each AI technician alternated the use of
semen from each bull; consequently, the technician effect observed in Experiment 1 may be related to semen handling and site of semen deposition. Body condition score tended to affect P/TAI (P ⫽ 0.09). Cows with BCS ⱖ 2.75 were 1.4 times more likely to become pregnant to first-service TAI compared with cows with BCS ⬍ 2.75. Furthermore, cows with BCS ⬍ 2.75 achieved a P/TAI eight percentage points less than cows with BCS ⱖ 2.75, (38% vs. 46%, respectively). Other studies have reported a similar P/TAI for suckled multiparous Nelore cows with BCS ⬍ 2.75 (42.5% [25]; 40.2% [26]). The decreased reproductive success of Nelore cows with low BCS at first-service TAI may be related to reduced basal LH levels [27], suppressed LH pulsatility [27], or both. Reduced pulsatile LH levels may be a consequence of increased sensitivity of the hypothalamus to estradiol negative feedback, resulting in reduced follicular growth, which may ultimately lead to anovulation [28]. Although the protocol used in the present study included 48-h calf removal, which has been shown to increase LH pulsatility [29 –31], Nelore cows with a lower BCS exhibited a lower P/TAI response than cows with a higher BCS, perhaps because the temporary weaning could not overcome the deleterious effect of inadequate nutrition (i.e., delay in LH response to calf removal as previously described [32]). 3.3. Experiment 2: fertility following TAI of nulliparous Nelore heifers Fertility, as measured by P/TAI, was different (P ⫽ 0.04) between FAA-positive and FAA-negative bulls (33.7% vs. 40.7%, respectively). These results were unexpected and contradict a previous report of increased fertility following AI with FAA-positive sperm [7]. Nevertheless, there is evidence of individual bulls with FAA-negative sperm achieving greater fertility at first AI service than individual bulls with FAA-positive sperm [7]. There was no effect of AI technician or BCS on P/TAI. It is known that the mere presence of FAA on spermatozoal membranes does not guarantee higher fertility [7]. With a limited number of bulls and TAI in both experiments presented here, it appears that FAAnegative status was not a limiting factor for fertility as measured by P/TAI. The same bulls and ejaculates were used for both experiments, and the relative fertility, within bull and across the two experiments appeared to be similar for five of the six bulls (Table 1). Bull C, however, exhibited an 18 percentage point difference in P/TAI between cows and heifers (45.8%
J.C. Dalton et al. / Theriogenology 77 (2012) 389 –394 Table 1 Fertility to first TAI in multiparous lactating Nelore cows and nulliparous Nelore heifers inseminated with either FAA-positive (⫹) or FAA-negative (⫺) sperm. Bull
A B C D E F
FAA status
⫹ ⫹ ⫹ ⫺ ⫺ ⫺
Experiment 1: cows
Experiment 2: heifers
Pregnant, N/total TAI (P/TAI %)
Pregnant, N/total TAI (P/TAI %)
51/127 (40.2) 55/142 (38.7) 60/131 (45.8) 57/145 (39.3) 59/145 (40.7) 55/145 (37.9)
44/112 (39.3) 32/98 (32.7) 25/90 (27.8) 37/104 (35.6) 52/120 (43.3) 40/93 (43.0)
FAA, fertility-associated antigen; P/TAI, pregnancy per TAI; TAI, timed artificial insemination.
vs. 27.8%, respectively). Consequently, caution must be used when interpreting these results, due to the small number of bulls used, and the observation that the differences in fertility observed in Experiment 2 among bulls exhibiting FAA-negative and FAA-positive sperm disappeared when bull C was removed from the statistical analysis. In conclusion, within the small number of bulls used, P/TAI was: (1) not different between FAA-positive and FAA-negative bulls in suckled multiparous Nelore cows; and (2) greater for FAA-negative bulls as compared with FAA-positive bulls following TAI in nulliparous Nelore heifers. Consequently, the identification of FAA-positive and FAA-negative status was unsuccessful as a method to evaluate the potential fertility of Nelore bulls to be used in TAI. Acknowledgments This research was supported in part by FMVZUNESP (Botucatu, SP, Brazil), Alta Genetics Brazil (Uberaba, MG, Brazil), and the University of Idaho (Caldwell, ID, USA). The authors thank Barbara Jackson of Animal Health Express (Tucson, AZ, USA) for donation of the ReproTest lateral flow cassettes, and the staff at Alta Genetics Brazil, Fazenda Anita (Mato Grosso do Sul, Brazil), and Agropecuária Fazenda Brazil (Barra do Garças, MT, Brazil), for their assistance in completing this research. References [1] Amann RP, Hammerstedt RH. In vitro evaluation of sperm quality: an opinion. J Androl 1993;14:397– 406. [2] Miller DJ, Winer MA, Ax RL. Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin. Biol Reprod 1990;42:899 –915.
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[3] Nass SJ, Miller DJ, Winer MA, Ax RL. Male accessory sex glands produce heparin-binding proteins that bind to caudal epididymal spermatozoa and are testosterone dependent. Mol Reprod Dev 1990;25:237– 46. [4] Bellin ME, Hawkins HE, Ax RL. Fertility of range beef bulls grouped according to presence or absence of heparin binding proteins in sperm membranes and seminal fluid. J Anim Sci 1994;72:2441– 8. [5] Bellin ME, Hawkins HE, Oyarzo JN, Vanderboom RJ, Ax RL. Monoclonal antibody detection of heparin-binding proteins on sperm corresponds to increased fertility of bulls. J Anim Sci 1996;74:173– 82. [6] Bellin ME, Oyarzo JN, Hawkins HE, Zhang H, Smith RG, Forrest DW, et al. Fertility-associated antigen on bull sperm indicates fertility potential. J Anim Sci 1998;76:2032–9. [7] Sprott LR, Harris MD, Forrest DW, Young J, Zhang HM, Oyarzo JN, et al. Artificial insemination outcomes in beef females using bovine sperm with a detectable fertility-associated antigen. J Anim Sci 2000;78:795– 8. [8] Cancel AM, Chapman DA, Killian, GJ. Osteopontin is the 55-kilodalton fertility-associated protein in Holstein bull seminal plasma. Biol Reprod 1997;57:1293–1301. [9] Killian GJ, Chapman DA, Rogowski LA. Fertility-associated proteins in Holstein bull seminal plasma. Biol Reprod 1993;49: 1202–7. [10] Parent S, Lefiévre L, Brindle Y, Sullivan, R. Bull subfertility is associated with low levels of a sperm membrane antigen. Mol Reprod Dev 1999;52:57– 65. [11] Roncoletta M, da Silva Carvalho Morani E, Esper CR, Barnabe VH, Franceschini PH. Fertility-associated proteins in Nelore bull sperm membranes. Anim Reprod Sci 2006;91:77– 87. [12] McCauley TC, Dawson GR, Oyarzo JN, McVicker J, Marks SHF, Ax RL. Development and validation of a lateral-flow cassette for fertility diagnostics in bulls. In Vitro Diag Tech 2004;10:35–40. [13] DeJarnette JM. The effect of semen quality on reproductive efficiency. Vet Clin Food Anim Pract 2005;21:409 –18. [14] Barth AD, Oko RJ. Preparation of semen for morphological evaluation. In: Abnormal Morphology of Bovine Spermatozoa, Iowa University Press, 1989, pp. 8 –16. [15] Blom E. The ultrastructure of some characteristic sperm defects and a proposal for a new classification of the bull spermiogram. In: Atti del VII Simposio Int di Zootechnia Milan, 1972, pp. 125–39. [16] Blom E. Sperm morphology with reference to bull infertility. In: First All-India Symposium on Animal Reproduction, Ludhiana, 1977, pp. 66 – 81. [17] Houghton PL, Lemenager RP, Moss GE, Hendrix KS. Prediction of postpartum beef cow body composition using weight to height ratio and visual body condition score. J Anim Sci 1990; 68:1428 –37. [18] SAS Institute Inc. SAS 9.1.3. SAS Institute Inc., 2000. [19] Sprott LR, Novasod A, Galino J, Dawson GR, McCauley TC, Forrest D, et al. Case study: fertility-associated antigen in peripubertal beef bulls. Prof Anim Sci 2006;22:353–7. [20] DeJarnette JM, Marshall CE, Lenz RW, Monke DR, Ayars WH, Sattler CG. Sustaining the fertility of artificially inseminated dairy cattle: the role of the artificial insemination industry. J Dairy Sci 2004;87:E93–104. [21] Dias CC, Wechsler FS, Day ML, Vasconcelos JLM. Progesterone concentrations, exogenous equine chorionic gonadotropin, and timing of prostaglandin F2␣ treatment affect fertility in postpuberal Nelore heifers. Theriogenology 2009;72:378 – 85.
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Theriogenology 77 (2012) 389 –394 www.theriojournal.com
Fertility-associated antigen on Nelore bull sperm and reproductive outcomes following first-service fixed-time AI of Nelore cows and heifers J.C. Daltona,*, L. Deragonb, J.L.M. Vasconcelosc, C.N. Lopesd, R.F.G. Perese, A. Ahmadzadehf a
Department of Animal and Veterinary Science, University of Idaho, Caldwell, Idaho, USA b Alta Genetics, Uberaba, Minas Gerais, Brazil c Departamento de Produçáo Animal, Faculdade de Medicina Veterinária e Zootecnia-UNESP, Botucatu, São Paulo, Brazil d Fazenda Anita, Mato Grosso do Sul, Brazil e Agropecuária Fazenda Brazil, Barra do Garças, Mato Grosso, Brazil f Department of Animal and Veterinary Science, University of Idaho, Moscow, Idaho, USA Received 27 May 2011; received in revised form 29 July 2011; accepted 4 August 2011
Abstract The objective was to determine whether the presence of fertility-associated antigen (FAA) on sperm collected from Nelore (Bos indicus) bulls can be used to assess potential fertility of sperm for use at first-service fixed-time AI (TAI). Six Nelore bulls were selected based on FAA status (FAA-negative: N ⫽ 3; FAA-positive: N ⫽ 3) and the ability to produce neat semen with ⱖ 70% morphologically normal sperm and 60% estimated progressive motility before cryopreservation. In Experiment 1, suckled multiparous Nelore cows (N ⫽ 835) were evaluated for body condition score (BCS) and received an intravaginal progesterone device (CIDR) and 2.0 mg of estradiol benzoate (Day 0). On Day 9 the CIDR was removed, 12.5 mg of PGF2␣ and 0.5 mg of estradiol cypionate were administered, and calves were removed for 48 h. All cows received TAI on Day 11 (48 h after CIDR removal). Pregnancy per TAI (P/TAI) was not different between FAA-positive and FAA-negative bulls (41.5% vs. 39.3%, respectively). There was an effect of AI technician on P/TAI (36.0% vs. 43.9%; P ⬍ 0.05) and BCS tended to affect P/TAI (P ⫽ 0.09), as cows with BCS ⱖ 2.75 were 1.4 times more likely to become pregnant compared with cows with BCS ⬍ 2.75. In Experiment 2, nulliparous Nelore heifers (N ⫽ 617) were evaluated for BCS and received a CIDR and estradiol benzoate (2.0 mg) on Day 0. On Day 7, all heifers received PGF2␣ (12.5 mg). On Day 9, CIDR inserts were removed and all heifers received estradiol cypionate (0.6 mg) and 200 IU eCG. All heifers received TAI on Day 11 (48 h after CIDR removal). Pregnancy/TAI was different (P ⫽ 0.04) between FAA-positive and FAA-negative bulls (33.7% vs. 40.7%, respectively). Presence of FAA on sperm was unsuccessful in assessing the potential fertility of sperm for use in TAI. © 2012 Elsevier Inc. All rights reserved. Keywords: Sperm; Fertility-associated antigen; Fixed-time AI; Bos indicus; Cattle
1. Introduction
* Corresponding author. Tel.: ⫹1 208 459 6365; fax: ⫹1 208 454 7612. E-mail address: [email protected] (J.C. Dalton). 0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.08.011
Synchronization programs that include fixed-time artificial insemination (TAI) provide an organized and efficient approach to administering AI. Consequently, beef and dairy cattle producers throughout the world are interested in identifying the most fertile bulls for
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TAI. Although many sperm attributes necessary for fertilization are known, including normal morphology, progressive motility, capacity for hyperactive motility, acrosomal enzymes, and chromatin integrity, many other attributes remain unknown [1]. Heparin-binding proteins (HBP) are secreted by the seminal vesicles, prostate, and Cowper’s glands, and coat sperm during ejaculation [2,3]. In 1994, Bellin and coworkers [4] reported that the distribution of specific forms of HBP on sperm corresponded to fertility potential of bulls used for natural service. Different patterns of HBP on bovine sperm have since been described [5,6]. Bulls with detectable fertility-associated antigen (FAA), a 31kDa molecular weight protein on sperm, were 9 to 40 percentage points more fertile (following natural service) than bulls producing sperm lacking FAA [5,6]. Sprott et al. [7] used 25 bulls of mixed breeds, including Bos taurus, Bos taurus ⫻ Bos indicus, and Bos indicus (Brahman) to investigate whether FAA could be used to assess the potential fertility of sperm to be used for AI. Bulls with sperm that were FAApositive were 7 to 9 percentage points more fertile following first-service AI than bulls producing sperm lacking FAA [7]. Collectively, the results of Bellin et al. [5,6] and Sprott et al. [7] provided evidence that FAA appeared to be an important trait of sperm that may be used in the evaluation of fertility potential. Nevertheless, the use of sophisticated techniques to determine the presence of FAA [6,7] and other proteins [8 –11] associated with fertility is not practical outside the research laboratory. McCauley et al. [12], however, reported on the development and validation of a lateral flow cassette for the rapid determination of FAA status in bulls, without the requirement for time-consuming, intensive techniques that must be performed in a research laboratory. Taken together with the realization that identification of novel spermatozoal or seminal attributes associated with male fertility may increase the accuracy of fertility estimation [13], the objective of this research was to use the commercially available lateral flow cassette to help determine whether the presence of FAA on sperm collected from Nelore (Bos indicus) bulls can be used to assess potential fertility of sperm for use at first-service TAI in multiparous Nelore cows and nulliparous Nelore heifers. 2. Materials and methods 2.1. Bulls, semen collection, and determination of spermatozoal FAA status Ejaculates from Nelore bulls (N ⫽ 49; range in age: 2 yr 8 mo to 11 yr) housed at Alta Genetics, Uberaba,
MG, Brazil, were collected by artificial vagina. Each ejaculate was preceded by two false mounts separated by 2 min of restraint. Immediately after collection and determination of ejaculate volume, 2.0 mL of neat semen was removed for use in a lateral flow cassette (ReproTest, Midland Bioproducts Corporation, Boone, IA, USA) which facilitated rapid on-site determination of spermatozoal FAA status as described previously [12]. Six Nelore bulls were selected for the field trial, based on presence or absence of FAA (FAA-positive: N ⫽ 3; FAA-negative: N ⫽ 3, respectively), on the ability to produce neat semen with ⱖ 70% morphologically normal sperm, and 60% estimated progressive motility before cryopreservation. 2.2. Semen evaluation, extension, and cryopreservation Immediately after collection, the vial containing the ejaculate was placed in a water bath at 32 °C. Concentration was determined using a spectrophotometer calibrated for bovine sperm using a hemacytometer. Motility was estimated to the nearest 5% after viewing several microscopic fields at magnification 250 X using a phase contrast microscope equipped with a heated stage (37 °C). The neat semen and extender (AndroMed, Minitube, Tiefenbach, Germany) were maintained in the same water bath. The semen was diluted 1:1 with extender and remained in the water bath for not more than 10 min. Subsequently, the semen was fully extended to 120 ⫻ 106 sperm per mL, and passively cooled to 18 °C for approximately 1 h. To facilitate sperm morphologic analysis, wet smears of extended semen fixed with glutaraldehyde were prepared [14]. Sperm abnormalities were quantified using differential interference contrast microscopy at magnification 1200 X and oil immersion. Differential counts of 100 cells from each of two wet smears of the fixed sample were averaged. Specific cell abnormalities were classified as described by Blom [15,16]. The extended semen was packaged in 0.25-mL straws (30 ⫻ 106 sperm), and the straws were placed in a cooling cabinet at 5 °C for 3.5 h for equilibration. Semen was cryopreserved using a programmable freezer (DigiCool, Instruments de Medecine Veterinaire, l’Aigle, France) and was stored in liquid nitrogen (⫺196 °C) until use. For evaluation of progressive motility postthaw, sample straws were thawed in water at 37 °C for 1 min. Sperm motility was estimated immediately after thawing and after 3 h of incubation at 37 °C as previously described.
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2.3. Experiment 1: animals and fixed-time AI protocol Suckled multiparous Nelore cows (N ⫽ 835) 40 to 60 days postpartum, were evaluated for body condition score (BCS; one to five scale; [17]) and enrolled in a first-service TAI program. All cows were maintained on Brachiaria decumbens pasture at a commercial beef cattle ranch in Mato Grosso do Sul, Brazil, with ad libitum access to water and mineral during the experimental period. On Day 0, the synchronization protocol began with a new intravaginal insert containing 1.9 g progesterone (CIDR; Pfizer Animal Health, São Paulo, SP, Brazil; new CIDR: first use), a CIDR that had been used previously in a 9-day synchronization protocol (second use), or a CIDR that had been used in two previous 9-day synchronization protocols (third use), and estradiol benzoate (2.0 mg im; Estrogin, Farmavet, São Paulo, SP, Brazil). On Day 9, CIDR inserts were removed and all cows were treated with PGF2␣ (PGF; 12.5 mg im; Lutalyse, Pfizer Animal Health, São Paulo, SP, Brazil) and estradiol cypionate (0.5 mg im; ECP, Pfizer Animal Health). Immediately after CIDR withdrawal, calves were removed from cows for 48 h. During the period of calf removal, calves were held in pens out of sight from their dams, and had ad libitum access to water. On Day 11 (48 h after CIDR removal), all cows received TAI. Two experienced technicians performed the inseminations, with each technician using semen from each bull. To randomize semen, AI technicians alternated usage of one straw from each bull. Calves were reunited with their dams immediately after TAI. Pregnancy was diagnosed by transrectal ultrasonography 28 to 35 days after TAI. 2.4. Experiment 2: animals and fixed-time AI protocol Nulliparous Nelore heifers (N ⫽ 617; 24 to 28 mo of age; 300 to 340 kg body weight) were evaluated for BCS (one to five scale; [17]) and enrolled in a firstservice TAI program. All heifers were maintained on Brachiaria decumbens pasture at a commercial beef cattle ranch in Mato Grosso, Brazil, with ad libitum access to water and mineral during the experimental period. On Day 0, heifers began the synchronization protocol with a second-use (9-day previously used) intravaginal progesterone insert (CIDR; Pfizer Animal Health) and estradiol benzoate (2.0 mg im; Estrogin, Farmavet). On Day 7, all heifers were treated with PGF2␣ (12.5 mg im; Lutalyse, Pfizer Animal Health). On Day 9, CIDR inserts were removed and all heifers were treated with ECP (0.6 mg im; Pfizer Animal
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Health) and equine chorionic gonadotropin (200 IU im; Folligon; Intervet Schering-Plough, São Paulo, SP, Brazil). On Day 11, all heifers received TAI 48 h after CIDR removal. Two experienced technicians performed the inseminations, with each technician using semen from each bull. To randomize semen, AI technicians alternated usage of one straw from each bull. Pregnancy was diagnosed by transrectal ultrasonography 28 to 35 days after TAI. 2.5. Statistical analysis For both experiments, pregnancy per TAI (P/TAI) data were analyzed by logistic regression using SAS [18]. For multiparous cows (Experiment 1), the full statistical model included the effects of treatment (FAA-positive or FAA-negative), sire (six bulls), technician (two inseminators), BCS (⬍ 2.75 and ⱖ 2.75, on scale of one to five), CIDR (new or used), treatment by technician, treatment by BCS, and technician by BCS. All two-way interactions with a P value ⬎ 0.25 were eliminated from the model; hence independent variables included in the final model were treatment, sire, technician, BCS, and CIDR. For nulliparous heifers (Experiment 2), the full statistical model included the effects of treatment (FAA-positive or FAA-negative), sire (six bulls), technician (two inseminators), BCS (⬍ 3.0 and ⱖ 3.0, on scale of one to five), treatment by technician, treatment by BCS, and technician by BCS. All two-way interaction variables with a P value ⬎ 0.25 were eliminated from the model; hence independent variables included in the final model were treatment, sire, technician, and BCS. 3. Results and discussion 3.1. FAA status of Nelore bulls This was the first trial conducted using the commercially available lateral flow cassette for determination of FAA status and comparison of fertility following first-service TAI in multiparous and nulliparous Bos indicus cattle using cryopreserved semen. Fifty tests were conducted on ejaculates from 49 bulls. Fortyseven tests (on ejaculates from 47 bulls) provided a distinct determination of FAA status. Three tests on ejaculates from two bulls were inconclusive (due to failure of the test, as evidenced by lack of a visible control band on the cassette). Five bulls (11%; 5/47) were FAA-negative, whereas 42 bulls (89%; 42/47) were FAA-positive. The proportion of FAA-negative Nelore bulls (11%) was similar to a previous report
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(12% FAA-negative; [6]), but less than in other studies using Bos taurus, Bos taurus ⫻ Bos indicus, and Brahman (Bos indicus) bulls (28% FAA-negative, [7]; 22% FAA-negative, [19]). Caution must be used when interpreting FAA status of a group, however, as the range of FAA-negative bulls [6] was 0 to 50% (depending on the group of bulls tested). Furthermore, it is possible that the apparent differences between studies in average proportion of FAA-negative bulls may be related to intensity of selection for overall fertility, as 100% of Holstein bulls tested and housed at AI centers were FAA-positive [6], compared with 89% of Nelore bulls tested and housed at the AI center in the present study. In contrast, the bulls used by Sprott et al. [7,19] were not housed at an AI center; consequently, it is possible that less intense selection for fertility resulted in a greater proportion of FAA-negative bulls [7,19]. 3.2. Experiment 1: fertility following TAI of suckled multiparous Nelore cows Fertility, as measured by P/TAI, was not different between FAA-positive and FAA-negative bulls (41.5% vs. 39.3%, respectively). These results were not consistent with the beneficial effect of FAA-positive bulls on pregnancy rates (pooled) to first AI in beef cows [7]; however, these results agreed with the observation that some FAA-positive bulls yield pregnancy rates similar to those of FAA-negative bulls following AI [7]. Within the bulls used in the present study, FAA-negative status was not a limiting factor of fertility. Apparently, sperm from both FAA-positive and FAA-negative bulls possessed sufficient levels of known and unknown characteristics [1] necessary to achieve similar fertility. Given that the overall objective of an AI center quality control program is to minimize variation in sire fertility [20], that similar P/TAI resulted following the use of sperm (with and without detectable FAA) from bulls housed at an AI center was not entirely unexpected. There was no effect of type of CIDR (new: first-use, 9-days or 18-days previously used: second-use or thirduse, respectively) on P/TAI, which agrees with previous reports [21,22] investigating previously used CIDR inserts in synchronization protocols with Bos indicus cattle. There was an effect of AI technician on P/TAI (36.0% vs. 43.9%; N ⫽ 375 and N ⫽ 460 respectively; P ⬍ 0.05); however, there was no AI technician by FAA status (treatment) interaction. Previous reports of differences in AI technician success have been reported [23–25]. Cows entered the breeding chute in random order and each AI technician alternated the use of
semen from each bull; consequently, the technician effect observed in Experiment 1 may be related to semen handling and site of semen deposition. Body condition score tended to affect P/TAI (P ⫽ 0.09). Cows with BCS ⱖ 2.75 were 1.4 times more likely to become pregnant to first-service TAI compared with cows with BCS ⬍ 2.75. Furthermore, cows with BCS ⬍ 2.75 achieved a P/TAI eight percentage points less than cows with BCS ⱖ 2.75, (38% vs. 46%, respectively). Other studies have reported a similar P/TAI for suckled multiparous Nelore cows with BCS ⬍ 2.75 (42.5% [25]; 40.2% [26]). The decreased reproductive success of Nelore cows with low BCS at first-service TAI may be related to reduced basal LH levels [27], suppressed LH pulsatility [27], or both. Reduced pulsatile LH levels may be a consequence of increased sensitivity of the hypothalamus to estradiol negative feedback, resulting in reduced follicular growth, which may ultimately lead to anovulation [28]. Although the protocol used in the present study included 48-h calf removal, which has been shown to increase LH pulsatility [29 –31], Nelore cows with a lower BCS exhibited a lower P/TAI response than cows with a higher BCS, perhaps because the temporary weaning could not overcome the deleterious effect of inadequate nutrition (i.e., delay in LH response to calf removal as previously described [32]). 3.3. Experiment 2: fertility following TAI of nulliparous Nelore heifers Fertility, as measured by P/TAI, was different (P ⫽ 0.04) between FAA-positive and FAA-negative bulls (33.7% vs. 40.7%, respectively). These results were unexpected and contradict a previous report of increased fertility following AI with FAA-positive sperm [7]. Nevertheless, there is evidence of individual bulls with FAA-negative sperm achieving greater fertility at first AI service than individual bulls with FAA-positive sperm [7]. There was no effect of AI technician or BCS on P/TAI. It is known that the mere presence of FAA on spermatozoal membranes does not guarantee higher fertility [7]. With a limited number of bulls and TAI in both experiments presented here, it appears that FAAnegative status was not a limiting factor for fertility as measured by P/TAI. The same bulls and ejaculates were used for both experiments, and the relative fertility, within bull and across the two experiments appeared to be similar for five of the six bulls (Table 1). Bull C, however, exhibited an 18 percentage point difference in P/TAI between cows and heifers (45.8%
J.C. Dalton et al. / Theriogenology 77 (2012) 389 –394 Table 1 Fertility to first TAI in multiparous lactating Nelore cows and nulliparous Nelore heifers inseminated with either FAA-positive (⫹) or FAA-negative (⫺) sperm. Bull
A B C D E F
FAA status
⫹ ⫹ ⫹ ⫺ ⫺ ⫺
Experiment 1: cows
Experiment 2: heifers
Pregnant, N/total TAI (P/TAI %)
Pregnant, N/total TAI (P/TAI %)
51/127 (40.2) 55/142 (38.7) 60/131 (45.8) 57/145 (39.3) 59/145 (40.7) 55/145 (37.9)
44/112 (39.3) 32/98 (32.7) 25/90 (27.8) 37/104 (35.6) 52/120 (43.3) 40/93 (43.0)
FAA, fertility-associated antigen; P/TAI, pregnancy per TAI; TAI, timed artificial insemination.
vs. 27.8%, respectively). Consequently, caution must be used when interpreting these results, due to the small number of bulls used, and the observation that the differences in fertility observed in Experiment 2 among bulls exhibiting FAA-negative and FAA-positive sperm disappeared when bull C was removed from the statistical analysis. In conclusion, within the small number of bulls used, P/TAI was: (1) not different between FAA-positive and FAA-negative bulls in suckled multiparous Nelore cows; and (2) greater for FAA-negative bulls as compared with FAA-positive bulls following TAI in nulliparous Nelore heifers. Consequently, the identification of FAA-positive and FAA-negative status was unsuccessful as a method to evaluate the potential fertility of Nelore bulls to be used in TAI. Acknowledgments This research was supported in part by FMVZUNESP (Botucatu, SP, Brazil), Alta Genetics Brazil (Uberaba, MG, Brazil), and the University of Idaho (Caldwell, ID, USA). The authors thank Barbara Jackson of Animal Health Express (Tucson, AZ, USA) for donation of the ReproTest lateral flow cassettes, and the staff at Alta Genetics Brazil, Fazenda Anita (Mato Grosso do Sul, Brazil), and Agropecuária Fazenda Brazil (Barra do Garças, MT, Brazil), for their assistance in completing this research. References [1] Amann RP, Hammerstedt RH. In vitro evaluation of sperm quality: an opinion. J Androl 1993;14:397– 406. [2] Miller DJ, Winer MA, Ax RL. Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin. Biol Reprod 1990;42:899 –915.
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