The effects of gonadotrophin releasing hormone in prostaglandin F 2α

The effects of gonadotrophin releasing hormone in prostaglandin F 2α -based timed insemination programs for beef cattle J. A. Small 1, J. D. Ambrose 2, W. P. McCaughey, D. R. Ward, W. D. Sutherland, N. D. Glover, and R. Rajamahendran 3 Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 148.251.232.83 on 04/05/18 1 Agriculture and Agri-Food Canada, Research Centre, PO Box 1000A, Brandon, MB, Canada R7A 5Y3; 2 Alberta Agriculture, Food and Rural Development, 6909-116th St, Edmonton, AB, Canada T6H 4P2; 3 University of British Columbia, Vancouver, British Columbia, Canada V6T 2A2. Received 25 July 2000, accepted 29 March 2001. Small, J. A., Ambrose D. J., McCaughey, W. P., Ward, D. R., Sutherland, W. D., Glover, N. D. and Rajamahendran, R. 2001. The effects of gonadotrophin releasing hormone in prostaglandin F 2α -based timed insemination programs for beef cattle. Can. J. Anim. Sci. 81: 335 343. Trials were conducted in the spring (May; n = 324) and fall (October; n = 132) with crossbred continental-type beef cows assigned on the basis of parity and postpartum interval to one of three timed-ai treatments and one of two post-ai treatments. The timed-ai treatments were: (DPG) double (14 d apart) (Lutalyse ) and AI (day = 0) 72 h after the second (day 3); (OVS) Ovsynch with the second GnRH (Factrel ) at 48 h and AI at 66 h; and (BRC) the same as OVS except that the second GnRH was given at the time of AI. Half of the cows within each treatment were given GnRH on day 14. Plasma progesterone concentrations were determined for the day of the first injection and on days 3, 0, 14, and 21. Timed-AI pregnancy was diagnosed by ultrasonography at day 42 and confirmed at calving. For DPG, OVS and BRC, responder rates were 75.9, 51.4 and 71.3%, respectively, in spring (P < 0.05) and 70.4, 70.4 and 59.1% in fall (P > 0.05), and AI pregnancy rates were 28.7, 44.9 and 44.4% in spring (P < 0.05) and 25.0, 40.9 and 43.2% in fall (P > 0.05). Post-AI GnRH had no significant effect on pregnancy or conception rates or day 21 progesterone. The use of GnRH in the based timed-ai program improved pregnancy rates and the BRC treatment was as effective as OVS. Neither postpartum interval nor initial progesterone concentration influenced (P > 0.05) the effect of GnRH on AI pregnancy rate, and GnRH had no effect (P > 0.05) on twinning rate or gender ratio. Key words: Beef cows, estrous synchronization, pregnancy, timed-ai, progesterone Small, J. A., Ambrose, J. D., McCaighey, W. P., Ward, D. R., Sutherland, W. D., Glover, N. D. et Rajamahendran, R. 2001. Incidence de la gonadolibérine sur les programmes d insémination synchronisée avec la prostaglandine F 2α chez les vaches à viande. Can. J. Anim. Sci. 81: 335 343. Au printemps (mai; n = 324) et à l automne (octobre; n = 132), les auteurs ont entrepris des essais sur des vaches à viande hybrides de type continental réparties dans trois groupes selon le rang de portée et l intervalle post-partum. Chaque groupe était associé à un de trois traitements d IA synchronisée et à un de deux traitements après l IA. Les traitements d IA synchronisée étaient les suivants : double injection de PGF2α (Lutalyse ) à 14 jours d intervalle puis IA (jour 0) 72 h après la deuxième injection (jour 3) (traitement DPG); administration de Ovsynch avec la deuxième dose de GnRH (Factrel ) à 48 h et IA à 66 h (traitement OVS); et traitement OVS avec administration de la deuxième dose de GnRH au moment de l IA (traitement BRC). La moitié des animaux de chaque groupe ont reçu de la GnRH le 14 e jour. La concentration de progestérone dans le sang a été déterminée le jour de la première injection puis les jours -3, 0, 14 et 21. La fécondation par IA a été établie par échographie le 42 e jour et confirmée au vêlage. La proportion de vaches DPG, OVS et BRC qui réagissent à l administration de PGF2α s établissait respectivement à 75,9, à 51,4 et à 71,3 % au printemps (P < 0,05) et à 25,0, à 40,9 et à 43,2 % à l automne (P > 0,05). L administration de GnRH après l IA n a aucun effet significatif sur la fécondation ni le taux de conception ou la concentration de progestérone au 21 e jour. L administration de GnRH dans le cadre du programme d IA synchronisée avec la PGF2α augmente le taux de fécondation et le traitement BRC s avère aussi efficace que le traitement OVS. Ni l intervalle post-partum ni la concentration initiale de progestérone n influencent (P > 0,05) l action de la GnRH sur le taux de fécondation par IA et la GnRH n a aucune incidence (P > 0,05) sur le nombre de jumeaux et la proportion de rejetons des deux sexes. Mots clés: Vaches à viande, synchronisation de l œstrus, gravidité, IA synchronisée, progestérone Artificial insemination has not been widely adopted by the cow-calf industry because estrus detection is time-consuming (Odde 1990; Small and McCaughey 1999). Extensive management of beef herds makes estrus detection laborious, 1 To whom correspondence should be addressed: (e-mail: jsmall@em.agr.ca). 335 Abbreviations: AI, artificial insemination; BRC, modified Ovsynch timed-ai program; DPG, double timed-ai program; GnRH, gonadotrophin releasing hormone (Factrel ); OVS, Ovsynch timed-ai program; PGF, prostaglandin 2α (Lutalyse )

336 CANADIAN JOURNAL OF ANIMAL SCIENCE expensive and impractical and as a result the majority of beef cattle are pasture-bred by bulls. Timed-AI would be more attractive to producers if the procedure required minimal handling of cows, was cost-effective and, when combined with the use of bulls to breed cows that return to estrus, would result in a high overall pregnancy rate in a restricted breeding season. The efficacy of for synchronization of estrus is limited by the proportion of cows cycling at the beginning of the breeding season (Wiltbank 1970), day of the estrous cycle, and subsequent ovulation and function of the corpus luteum (King et al. 1982). Intramuscular injection of cows with GnRH has been shown to initiate growth of a new wave of follicles (Macmillan and Thatcher 1991; Twagiramungu et al. 1994), and/or to induce ovulation of a dominant follicle, resulting in the development of a new corpus luteum, and/or to enhance the function of an existing corpus luteum (Milvae et al. 1984). Therefore, GnRH has been used in synchronization programs for timed AI (Ovsynch ) to increase the proportion of cows that respond to and to synchronize ovulation of the ovulatory follicle (Pursley et al. 1997, 1998; Twagiramungu et al. 1995b). The timing of AI relative to expected ovulation has been reported to influence (Pursley et al. 1998; Wehner et al. 1997) or have no effect (Rorie et al. 1999) on gender ratio, and injection of GnRH around the time of insemination has been reported to increase the occurrence of twin ovulations (Tanabe et al. 1994). Post-AI progesterone supplementation using progesterone-releasing devices (Savio et al. 1993) or GnRH (Rettmer et al. 1992) or human chorionic gonadotropin (Rajamahendran and Sianangama 1992) have been used to enhance luteal function. These treatments have been reported to either increase or have no effect on pregnancy rates. The objectives of this study were to determine: (1) pregnancy and conception rates and plasma progesterone concentrations in beef cows following timed-ai in regimens requiring cows to be handled three vs four times; (2) if timed-ai pregnancy rates could be enhanced by an injection of GnRH around the time of maternal recognition of pregnancy; (3) overall pregnancy rates when cows are exposed to bulls for 40 d, starting 14 d after AI; (4) the effects of timed-ai on calving distribution, twinning and gender ratio. MATERIALS AND METHODS Treatments and Animals Two trials were conducted with crossbred beef cows, 324 in the spring (May) and 132 in the fall (October). Cows were assigned on the basis of parity and postpartum interval to one of three estrous synchronization treatments for timed-ai and one of two post-ai GnRH treatments. The timed-ai treatments were: (DPG) double (14 d apart) (25 mg i.m. doses of dinoprost; Lutalyse, Pharmacia & Upjohn, Orangeville, ON) and AI (day = 0) 72 h after the second (day 3); OVS (Ovsynch ), an initial injection of GnRH (100 µg i.m. Factrel, Ayerst Veterinary Laboratories, Guelph, ON) followed 7 d later by and a second injection of GnRH at 48 h with AI at 66 h after (18 h after GnRH); and (BRC) the same as OVS except that the second GnRH was given at the time of AI. Half of the cows within each treatment were given GnRH on day 14. All cows were inseminated with semen from a Gelbvieh sire with proven fertility. Return services were by fertile 3-yr-old Gelbvieh bulls, placed with the cows for 40 d starting on day 14. Bulls were evaluated for breeding soundness prior to the breeding season. The ratio of cows to bulls was 48:1 from 14 to 25 d after AI and 72:1 for the remainder of the breeding season. All handling and management of animals was in accordance with the guidelines of the Canadian Council on Animal Care (1993) and the Canadian Code of Practice for the care and handling of beef cattle (Agriculture Canada 1990). Spring Herd Management Trial 1 The spring herd consisted of suckled 2-yr-old (1st parity, n = 80), 3 year-old (2nd parity, n = 122) and mature (3+ parity, n = 122) cows. Calving occurred between 7 March and 7 May for multiparous cows, 15 March and 12 May for biparous cows, and 30 January and 12 May for primiparous cows. The interval from parturition to AI was 64 ± 21 d (range, 24 to 124 d). The primiparous cows used in this study were crosses of the Hereford and Simmental breeds or were Composite ( 1 4 Simmental 1 4 Charolais 1 16 Limousin 7 16 British) while all 2+ parity cows were Composite. Calves were progeny of British (Red Angus or Hereford) or Continental (Simmental, Composite or Gelbvieh) sires. The composition of the diets fed before and after calving is shown in Table 1. Cows were given corn-silage, a pelleted concentrate supplement and straw prior to calving; when cows moved to the calving facility, hay replaced straw. Heifers were given corn silage, a pelleted concentrate supplement and hay prior to and after calving. The pelleted supplement contained monensin (200 mg d 1, Rumensin, Elanco Animal Health, Guelph, ON) to control coccidiosis. Nutrient analysis of composite samples of corn silage, straw and hay were determined by near-infrared reflectance (Nor- West Laboratories, Winnipeg, MB). After AI, dams and their calves were turned out to either grass or grass-alfalfa pastures. All cows and heifers maintained optimum (moderate) body condition (Richards et. al. 1986) before and after calving. Mean body weight and condition score of cows at breeding were 590 ± 55 kg and 6.0 ± 0.4, respectively. Fall Herd Management Trial 2 The interval from parturition to AI was 64 ± 21 d (range, 24 to 124 d). The fall herd consisted of mature (3+ parity, n = 132) Composite cows, of which 28 were suckled (73 ± 10 d postpartum), and the remainder had calved in spring and had been weaned 30 d prior to AI. Calves were progeny of either Red Angus or Gelbvieh bulls. Cows were maintained on dormant perennial pasture (80% grass and 20% alfalfa) with trace-mineralized salt offered free-choice. All cows were inseminated on 30 October. All suckled cows maintained optimum (moderate) body condition (Richards et. al. 1986) before and after calving. Mean body weight and condition score of cows at breeding were 574 ± 67 kg and 6.1 ± 0.4, respectively.

SMALL ET AL. TIMED-AI OF BEEF COWS 337 Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 148.251.232.83 on 04/05/18 Table 1. Composition of diets for lactating beef cows in Spring (Trial 1) Item Corn silage Pellets z Straw y Hay x Amount offered (kg hd 1 d 1 ) 10.0 2.0 Free choice Free choice Dry matter (g kg 1 as fed) 371.0 870.0 856.0 839.0 Digestible energy (MJ) 12.9 13.4 6.4 10.1 Protein (g) 80.7 164.0 30.0 125.0 Neutral detergent fibre (g) 474.6 197.0 797.0 540.0 Acid-detergent fibre (g) 257.7 6.7 535.0 387.0 Calcium (g) 2.1 5.9 2.4 12.1 Phosphorus (g) 2.6 8.5 1.1 1.7 Potassium (g) 12.6 7.8 17.7 17.4 Magnesium (g) 1.9 4.1 1.0 2.3 z Pellets contained per MT: barley 851.4 kg, 44% protein soybean meal 115.0 kg, trace mineral 25.0 kg, cobalt-iodized salt 5.0 kg, vitamins A (10 000 KIU), D 3 (1000 KIU), E (100 KIU) 2.0 kg, Rumensin (monensin 200 mg kg 1 ) 0.74 kg, molassaid 0.5 kg. Trace mineral contained per kg: 155 g calcium, 155 g phosphorus, 120 g magnesium, 10 g potassium, 0.2 g iodine, 5 g iron, 4 g copper, 5 g manganese, 10 g zinc, 0.05 g cobalt, 2 g fluorine, 0.03 g selenium, 500 KIU vitamin A, 55 KIU vitamin D 3, 0.5 KIU vitamin E. Barley straw fed to mature cows (3rd parity and over) prior to calving. x Hay fed to 1st and 2nd parity cows before calving and all cows after calving. Blood Sampling and Progesterone Analysis Blood samples were collected for progesterone analysis at the time of initial hormone injections and on days 3, 0, 14 and 21. All blood samples were collected into 10 ml heparinized evacuated tubes by venipuncture of the coccygeal vein or artery. Plasma progesterone concentrations were determined by an established enzyme-immunoassay (Del Vecchio et al. 1995) which had a detection limit of 0.031 ng ml 1 and intra- and inter-assay coefficients of variation of 5.4 and 10.2%, respectively. Ovarian function was defined by plasma progesterone concentrations. A progesterone concentration greater than or equal to 1.0 ng ml 1 was considered high and indicative of a functional corpus luteum whereas progesterone less than 1.0 ng ml 1 was considered low and indicative of the absence of a functional corpus luteum. Only cows with high progesterone at the time of the day 3 and low progesterone at AI (day 0) were considered respondent. Non-respondents included asynchronous (high progesterone at AI) or non-cycling (low progesterone throughout synchronization program). Response to the initial injections of either GnRH or was also defined in terms of progesterone concentration. Respondents were defined as either low or high progesterone initially and high progesterone at the time of (day 3). Pregnancy Diagnosis Timed-AI pregnancy was diagnosed by real-time transrectal ultrasonography (Aloka 560, 5.0 MHz probe, Aloka, Japan) on day 42. At this time, ultrasonography could be used to accurately distinguish a cow pregnant to timed-ai versus a cow that was pregnant to natural service (Kastelic et al. 1988). On day 90, pregnancies achieved over the entire breeding season (AI and natural service) were diagnosed by rectal palpation. Presumptive AI pregnancy (day 21) was defined as low progesterone on day 0 and high progesterone on day 14 and 21. Timed-AI conception rate was defined as the proportion of AI-pregnant respondents. Apparent embryonic loss was defined as the difference between pregnancy (day 42) and presumptive (day 21) pregnancy. Statistical Analysis Categorical Models (CATMOD) procedure of the Statistical Analysis System p.c. v 6.12 (SAS Institute, Inc. 1988) were used to perform maximum likelihood chi-square analysis of variance to test differences in proportions. The main effects in the model were timed-ai program (T), post-ai GnRH treatment (PA) and the interaction. Further statistical analyses were conducted to explore factors that may have influenced synchronization, responder, and AI conception rates. The SAS univariate (UNIVARIATE) procedure was used to define categories of postpartum interval with an approximately equal number of observations within timed-ai and post-ai GnRH treatments. Three postpartum categories were identified; < 60 d, 60 to 120 d and dry. The model of pooled data for the CAT- MOD procedure included postpartum category, timed-ai treatment, post-ai GnRH treatment and all two- and threeway interactions. Pearson correlation coefficients were generated using SAS Frequency procedures. Data for one 2nd parity cow in the spring trial with health problems unrelated to the experimental procedures were excluded from the data analysis. RESULTS AI Pregnancy and Calving Rates Timed-AI (day 0) pregnancy based on progesterone (day 21) and ultrasonography (day 42), apparent embryonic loss, cumulative pregnancy (AI and natural service) based on palpation (day 90), and calving rates for the spring and fall trials are shown in Table 2. Timed-AI pregnancy rates were: (1) higher with than without the use of GnRH in the synchronization program; (2) similar between the BRC and OVS methods; and (3) unaffected by day 14 post-ai GnRH. Apparent embryonic loss was lower with than without the use of GnRH in the synchronization program, similar between BRC and OVS methods, and unaffected by day 14 post-ai GnRH. Presumptive AI pregnancy, cumulative pregnancy and calving rates did not differ among synchronization treatments and were not affected by day 14 post-ai GnRH.

338 CANADIAN JOURNAL OF ANIMAL SCIENCE Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 148.251.232.83 on 04/05/18 Table 2. Timed-AI (day 0) pregnancy based on progesterone (day 21) and ultrasonography (day 42), apparent embryonic loss, cumulative pregnancy (AI plus natural service day 14 to day 54) based on palpation (day 90), calving, twinning and female birth rates (%) of beef cows inseminated after with or without GnRH treatment 14 d post-ai Timed-AI treatment z (T) d14 post-ai GnRH (PA) Probability y Calving distribution for the spring and fall herds is shown in Fig. 1. The interval (mean ± standard deviation) from AI to calving was 285 ± 5.9 (spring) and 286 ± 5.5 d (fall) for cows that conceived to AI and 314 ± 11.4 (spring) and 313 ± 10.1 d (fall) for bull-bred cows. For the spring and fall herds, calving began as early as 12 d before a 283 d predicted due date and for the cows that conceived to AI, 50% of calves were born within 7 d. The number of days from first to last calf was 21, 24 and 19 for DPG, OVS and BRC cows that conceived to timed-ai and 38 d for bull-bred cows There was no effect (P > 0.05) of GnRH on the occurrence of twin births or gender ratio (Table 2). For the spring herd, the occurrence of twin births for timed-ai was 3.7 (1/27), 4.9 (2/41), 4.9% (2/41) for DPG, OVS and BRC, respectively, and 1.5% (2/133) for bull-bred cows. For the fall herd, the occurrence of twins was 0 for timed-ai and 6.1% (4/65) for bull-bred cows. The proportion of female calves also did not differ (P > 0.05) between the spring and fall herds and combined for timed-ai was 43.5% (17/39), 55.4% (36/65) and 50.7% (32/59) for DPG, OVS and BRC, respectively and 52.9% (108/204) for bull-bred cows. Responder and AI Conception Rates The proportion of cows with high progesterone initially and on day 3, responder and AI conception rates are shown in Table 3. The proportion of cows with high progesterone initially did not differ (P > 0.05) among treatment groups in either spring or fall. The proportion of cows with high progesterone on day 3 and respondent to was DPG OVS BRC SE No Yes SE T PA Spring herd (Trial 1) Number of cows 107 108 108 162 161 Presumptive AI pregnancy (day 21) x 55.6 54.2 52.8 3.7 53.7 54.7 2.8 0.919 0.864 AI pregnancy (day 42) 28.7a 44.9b 44.4b 3.7 51.2 40.0 2.7 0.047 0.767 Apparent embryonic loss w 26.8a 14.0b 10.2b 3.2 16.7 17.4 2.4 0.004 0.916 Cumulative pregnancy (day 90) 85.8 91.6 86.1 4.2 85.8 87.6 3.1 0.138 0.639 Calving v 94.9 98.8 98.8 3.2 75.3 74.5 2.6 0.159 0.907 Twin births u 3.7 4.9 2.4 1.6 3.3 Female calves born 49.3 56.8 51.7 4.0 58.9 46.8 2.9 0.610 0.063 Fall herd (Trial 2) Number of cows 44 44 44 66 66 Presumptive AI pregnancy (day 21) x 63.6 52.3 59.1 4.7 53.0 69.7 3.4 0.098 0.523 AI pregnancy (day 42) 25.0 40.9 43.2 4.6 31.8 40.9 3.4 0.115 0.278 Apparent embryonic loss w 38.6a 13.6b 15.9b 4.2 22.7 22.7 3.2 0.008 0.727 Cumulative pregnancy (day 90) 88.6 88.6 90.9 3.7 90.9 87.9 2.7 0.887 0.679 Calving v 100.0 97.4 95.0 3.8 98.3 96.6 2.8 0.943 0.679 Twin births u 0 2.6 7.9 5.1 1.8 0.400 Female calves born 46.1 48.7 60.9 4.8 58.1 45.6 3.6 0.326 0.153 z DPG: two doses of (Lutalyse ) given 14 d apart and AI at 72 h after the second dose; OVS (Ovsynch ); BRC same as OVS except that the second GnRH is given at the time of AI at 66 h after. Interaction among T and PA groups were not significant (P > 0.05); differences between trials were not significant (P > 0.05). x Progesterone low (< 1 ng ml 1 ) on day 0 and high on day 14 and day 21. w Difference between day 21 and day 42 AI pregnancy rate. v Proportion of cows pregnant at 90 d (retained) that calved, refer to text. u Insufficient observations for statistical analysis. a b Means with different letters are different (chi-sq P < 0.05). similar between DPG and BRC in the spring and fall, lowest for OVS in the spring, and highest for OVS in the fall (P < 0.05). In both trials, AI conception rates were highest (P < 0.05) for BRC, lowest for DPG and intermediate for OVS (P > 0.05). Progesterone concentrations, responder rates and AI conception rates did not differ (P > 0.05) between day 14 post-ai groups. Postpartum Interval, Initial Progesterone and Synchronization Rate To further explore factors affecting AI pregnancy rate, the data for the spring and fall trials were pooled and stratified by postpartum interval: < 60 d (n = 147), 60 + d (n = 202) and dry (n = 106). Postpartum interval did not influence (P > 0.05) AI pregnancy or the effectiveness of using GnRH in the -based timed-ai program. However, the proportion with high progesterone initially (36.0, 51.9 and 68.9%) and on day 3 (59.2, 73.3 and 78.3%) and responder rate (56.5, 68.8 and 66.0%) was lowest for cows < 60 d postpartum and similar between 60 + d and dry (P < 0.05). This indicated that initial progesterone rather than postpartum interval per se influenced synchronization rate. Therefore, the data were stratified by initial progesterone level for spring and fall (Table 4). The day 14 post-ai GnRH was excluded from the final statistical model because there had been no effect on AI pregnancy rates. The synchronization rate differed among treatments only when progesterone was high initially in spring (P < 0.05)

SMALL ET AL. TIMED-AI OF BEEF COWS 339 Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 148.251.232.83 on 04/05/18 Table 3. The proportion (%) of cows with high serum progesterone initially and on day 3, respondent to prostaglandin-f ( 2α ) on day 3 and conception rate (%) of beef cows inseminated (day 0) at a fixed time after with or without gonadotrophin releasing hormone (GnRH) treatment 14 d post-ai but not fall (P > 0.05). The responder rate was correlated with synchronization rate; the responder rate for cows with high progesterone on day 3 was 95.3% in spring (n = 323; r = 0.93; P < 0.001) and 84.6% in fall (n = 132; r = 0.73; P < 0.001). Initial progesterone did not influence (P > 0.05) the effect of GnRH on AI conception in spring or fall. AI pregnancy rate in synchronized cows was correlated with AI conception in spring (r = 0.99; P < 0.001) and fall (r = 0.98; P < 0.001). With GnRH in the timed-ai program, a number of asynchronous cows conceived to AI in spring (31.3%, n = 83) and fall (21.0 %, n = 19), in contrast to no GnRH in spring (7.6% n = 26) and fall (22.2% n = 9). Timed-AI treatment z (T) d14 post-ai GnRH (PA) Probability y DPG OVS BRC SE No Yes SE T PA Spring herd (Trial 1) Number of cows 108 107 108 162 161 High progesterone initially (%) 41.7 44.9 45.4 3.7 46.9 41.0 2.7 0.840 0.285 High progesterone day 3 (%) 75.9a 51.4b 71.3a 3.6 65.4 67.1 2.7 0.001 0.754 responders (%) x 72.2a 51.4b 65.7ab 3.7 64.1 72.5 2.7 0.005 0.761 AI conception (day 42%) w 37.2a 52.7ab 56.3b 3.6 50.5 45.6 2.7 0.001 0.487 Fall herd (Trial 2) Number of cows 44 44 44 66 66 High progesterone initially (%) 72.7 72.7 56.8 4.5 65.1 69.7 3.3 0.098 0.523 High progesterone day 3 (%) 79.6 88.6 68.2 4.2 80.3 77.2 3.1 0.063 0.670 responders (%) x 70.4 70.4 59.1 4.6 68.1 65.1 2.7 0.377 0.681 AI conception (day 42%) w 29.0a 48.3ab 65.3b 5.1 42.2 51.1 3.8 0.026 0.401 z Refer to Table 2. y Interaction among T and PA groups was not significant (P > 0.05). x High progesterone on day 3 and low progesterone day 0. w AI pregnancy rate (day 42) for respondents. a b Timed-AI treatment means with different letters are different (chi-sq P < 0.05). Table 4. The influence of high or low plasma progesterone (when based timed-ai treatments were initiated) on the synchronization, responder, AI conception and pregnancy rates (%) in beef cows Initial serum progesterone level (I) High Low Timed-AI treatment z (T) Probability DPG OVS BRC DPG OVS BRC SE I T I T Spring herd (Trial 1) Number of cows 45 48 49 63 59 59 Synchronization 97.7a 43.7c 65.3b 60.3b 57.6b 76.3b 4.2 0.057 <0.001 <0.001 responder 91.1a 43.7b 59.2b 58.7b 57.6b 71.2b 4.3 0.277 <0.001 <0.001 AI conception 34.1a 61.9b 62.1b 40.5a 47.1b 52.4b 4.9 0.411 0.036 0.430 AI pregnancy synchronous y 31.8a 61.9b 59.4b 39.5a 47.1b 48.9b 4.8 0.424 0.032 0.356 AI pregnancy asynchronous x 0 37.0 17.6 8.0 36.0 28.6 Fall herd (Trial 2) Number of cows 32 32 25 12 12 19 Synchronization 75.0 93.7 76.0 91.7 75.0 57.9 5.6 0.483 0.083 0.132 responder 62.5 78.1 64.0 91.7 50.0 52.6 5.8 0.921 0.213 0.056 AI conception 20.0a 48.0ab 62.5b 45.4a 50.0ab 70.0b 7.0 0.279 0.049 0.620 AI pregnancy synchronous y 16.7 40.0 52.6 41.7 41.7 36.8 6.7 0.130 0.092 0.511 AI pregnancy asynchronous x 25.0 50.0 33.3 0 33.3 0 z Refer to Table 2. High progesterone day 3. x Low progesterone day 3; insufficient observations for ANOVA. a b Timed-AI treatment means with different letters are different (chi-sq P < 0.05). DISCUSSION Timed-AI, Calving Distribution, Twinning and Gender Ratio This study and others (Pursley et al. 1995, 1997; Twagiramungu et al. 1995b; Roy and Twagiramungu 1997; Geary et al. 1998) have reported acceptable AI pregnancy rates (without the need for estrus detection), when GnRH was used in -based synchronization programs for cattle. The AI pregnancy rates achieved using GnRH were similar between the BRC and OVS methods, indicating that injection of a second dose of GnRH at the time of AI (BRC) at 66 h after was equally as effective as 18 h before

340 CANADIAN JOURNAL OF ANIMAL SCIENCE Fig. 1. The effect of timed-insemination of beef cows on calving distribution within the spring (a) and fall (b) herds. The start of the calving season (day 0 = 283 d from AI) was 16 March and 9 August for spring and fall, respectively. AI (OVS). Therefore, in agreement with others (Twagiramungu et al. 1995b; Geary and Whittier 1998), in beef cattle there is little advantage from administering the second GnRH separately from the time of AI. Thus, a threestep as opposed to a four-step timed-ai program may be an attractive option for synchronized breeding in commercial beef herds because of the reduced labor requirement. However, the timing of AI and the second GnRH and the pregnancy rates achieved differ among these studies as does the management superimposed and the products used. The impact of the timed AI programs on calving distribution is clearly shown in Fig 1. Although average calving dates were not significantly different among the timed-ai programs, the BRC method appeared to result in a more compact calving season. This may have been due to cows in the OVS group having a prolonged cycle when timed-ai failed. A prolonged cycle suggests that embryonic death preceded luteal regression (Kastelic et al. 1991b). Injection of GnRH at the time of insemination (12 h after estrus) has been reported to increase the occurrence of twin ovulations (Tanabe et al. 1994). Induction of twin ovulations may explain why there were a few more twin births from the GnRH timed-ai than DPG or bull-service in the spring but, there were no timed-ai twins in the fall. Therefore, the risk of GnRH-induced twin ovulations is not likely to pose a management problem. The timing of AI relative to expected ovulation has been reported to influence the gender ratio (Wehner et al. 1997; Pursley et al. 1998); heifer calves predominate from early insemination (20 h before ovulation) and bull calves predominate from late insemination (10 h before ovulation). Others have shown no effect of timing AI on gender ratio (Rorie et al. 1999). These studies involved less than 20 to 48 calves born per treatment and may have been influenced by the use of different sires and the assumption that ovulation in cows and heifers occurs at a similar time following estrus. Ovulation following AI was expected to occur 6 to 14 h later for OVS and 6 to 32 h later for BRC (although a surge in luteinizing hormone may have occurred before GnRH in some cows). The influence of sire was eliminated because, within each herd, only one sire was used for AI. No effect on gender ratio was detected and therefore it is unlikely that the timing of AI relative to ovulation would be a reliable way to influence the gender ratio. Effect of GnRH in -based Timed-AI Programs Overall, timed-ai pregnancy rate for DPG was low (28%), but this was not due to low responder rates (72%) or to abnormally short cycles since presumptive AI pregnancy rates were high (58%). Beef cows have been reported to show estrus 57 or 67 h after given in early (days 5 9) or late (days 10 15) diestrus (King et al. 1982; Berardinelli and Adair 1989). Therefore, timing AI at 72 h after was expected to be optimal (15 to 5 h after estrus) for DPG respondents. Although synchronization and responder rates were highest when progesterone was high initially, AI pregnancy and conception did not differ between high and low initial progesterone groups (Table 4). When progesterone was high both initially and on day 3, it can be deduced that the initial injection was administered between day 12 and day 17 of the estrous cycle and the second injection in the early- to mid-luteal phase (day 5 to day 11). In contrast, when progesterone was low initially and high on day 3, the second injection was likely administered in mid-cycle (day 10 to day 14). Generally, a wave of follicular growth is initiated on day 0 and 10, or day 0, 9 and 16 following ovulation in cows with two- or three-wave estrous cycles, respectively (Ginther et al. 1989). With administered in mid-cycle, in comparison to earlyluteal phase, the decline in blood progesterone is delayed by 8 h (32 h vs. 24 h) and ovulation is delayed by 36 h (108 vs. 72 h), respectively (Berardinelli and Adair 1989; Kastelic et al. 1991a). Therefore, AI at 72 h may have been from 16 to 36 h before ovulation and within an interval that spermatozoa (and to a lesser extent ova) remain viable in the female reproductive tract (Hafez 1987). With treatment in the early luteal phase (day 5 to day 8), ovulation of a 1st wave dominant follicle may have occurred 3.0 to 3.5 d later (Kastelic et al. 1991a) when the dominant follicle was 8 to 11 d of age at AI. In mid-cycle (day 12) a 2nd wave domi-

nant follicle may have ovulated 4.5 d later (Kastelic et al. 1991a) at 5 to 7 d of age at AI. From day 9 to day 11, the dominant follicle may have been in static or regressing phases from the 1st wave, absent or in the growing phase of the 2nd wave (Ginther et al. 1989) and, therefore, either 12 to 14 d or 3 to 5 d of age at AI. Therefore, in the present study with high synchronization and responder rates, poor AI pregnancy rates for DPG was likely primarily due to the failure to synchronize growth of the ovulatory follicle. Overall, including GnRH in the synchronization program improved -based timed-ai pregnancy rates 57% (28 vs. 44%), but this was not due to an increase in the proportion of cows that responded to, as reported by others (Pursley et al. 1998). However, responder rate was correlated with the proportion of cows with high progesterone on day 3, indicating a high degree of synchrony on day 3 since is less effective in the early luteal phase (King et al. 1982). The first injection of GnRH did not increase the proportion of cows with high progesterone on day 3 (Table 3) and did not increase synchronization rate (Table 4). In cows with high progesterone initially (presumably days 5 to 18 of the estrous cycle) the first injection of GnRH failed to maintain a corpus luteum in 45% of cows (44/97) in the spring and 14% (8/57) of cows in the fall. This may have been a seasonal, lactational, dietary or stage of the estrous cycle influence on the ovarian response to GnRH. Others have reported that GnRH given in mid-luteal phase prolonged the lifespan of the corpus luteum or partially protected the corpus luteum from spontaneous regression (Milvae et al. 1984; Twagiramungu et al. 1994). In theory, GnRH may prevent regression of the corpus luteum by causing the release of luteinizing hormone, which stimulates progesterone production (Milvae et al. 1984). However, it is the pulsatile release of luteinizing hormone during early to mid-luteal phase that maintains the corpus luteum during the estrous cycle (Peters et al. 1994). Therefore, a single surge or pulse of luteinizing hormone resulting from GnRH is not likely to effectively maintain the corpus luteum. In cows with low progesterone initially (presumably acyclic or days 0 to 5 of the estrous cycle) GnRH failed to induce a corpus luteum in 33% (n = 118) of cows in the spring and 35.5% (n = 31) in fall, consistent with previous work in dairy cattle (Vasconceles et al. 1999). Ovulation in response to GnRH varies by day of the estrous cycle (Twagiramungu et al. 1994; Vasconceles et al. 1999), but generally it has been reported that follicles greater than 10 mm in diameter ovulate following GnRH treatment (Crowe et al. 1993; Pursley et al. 1995). Vasconceles et al. (1999) reported an initial injection of GnRH induced ovulation in 64% of cows but varied from 23% on day 1 to 4 (day 0 = estrus), 96% day 5 to day 9, 54% day 10 to day 16, and 77% day 17 to day 21. These results are consistent with waves of follicular growth. Selection of a dominant follicle generally occurs within 3 d of wave emergence (Ginther et al. 1989), and in most cases it acquires ovulatory capacity after the expression of luteinizing hormone receptors in granulosa cells. Therefore, the high responder rate likely resulted primarily from spontaneous ovulation or existing corpora lutea. SMALL ET AL. TIMED-AI OF BEEF COWS 341 In this study, the advantage of using GnRH was more likely due to one or more of the following factors: ensuring the emergence of a new ovulatory follicle: ensuring that ovulation occurred at a more appropriate time relative to AI; or induction of accessory corpora lutea. Firstly, intramuscular injection of cows with GnRH has been shown to initiate growth of a new wave of follicles (Macmillan and Thatcher 1991; Twagiramungu et al. 1994) followed by (within 6 d of treatment) emergence and selection of a dominant follicle that becomes preovulatory following. A follicle that becomes preovulatory within 9 d of emergence has been considered the most fertile (Savio et al. 1993). In the present study, the first GnRH was expected to result in the emergence of an ovulatory follicle that was 9 d old when cows were inseminated and the second GnRH was expected to synchronize ovulation (Pursley et al. 1998) at 8 d for OVS and 9 d for BRC. The timing of AI relative to was 66 h for the OVS and BRC programs. Therefore, any difference between these treatments was expected to result from the timing of the second GnRH. The second GnRH at 48 h after has been shown to induce ovulation in over 80% of cows, regardless of whether or not the first GnRH induced ovulation (Vasconceles et al. 1999; Stevenson 2000). In dairy cattle, AI at 0, 8, 16, 24, and 32 h after the second GnRH (48 h after ) had a quadratic effect on conception; conception rates were highest from AI at 8 to 24 h after GnRH and lowest at 32 h (Pursley et al. 1998). Timing of AI would depend upon how well cows were synchronized, since ovulation of the first-wave dominant follicle occurs earlier than a second-wave dominant follicle following (Kastelic et al. 1991a). Although AI pregnancy rate did not differ between BRC and OVS, AI conception rate was highest for BRC, especially in fall. Some BRC cows may have ovulated before the second GnRH, but it is also possible that delaying GnRH to 66 h in beef cattle may have allowed the dominant follicle to acquire ovulatory capacity, especially in fall. In dairy cattle, the Ovsynch protocol has been less effective when initiated in late luteal phase because ovulation occurred either before the second GnRH or the second GnRH failed to induce ovulation, likely because the dominant follicle had not acquired ovulatory capacity (Vasconceles et al. 1999). Initiation of the Ovsynch protocol early in the estrous cycle has resulted in high synchronization but low AI pregnancy rates, possibly due to the presence of large preovulatory follicles (> 14 mm) at the time of (Vasconceles et al. 1999). In dairy cattle, milk production is negatively correlated with serum progesterone and fertility and positively correlated with follicular size (Vasconceles et al. 1999). Low progesterone concentrations are believed to result in larger follicles because of an increase in pulsatile luteinizing hormone. The suckled beef cows in our study were in good body condition, on a rising plane of nutrition throughout the breeding season, and in Fall the majority of cows were dry. Therefore, milk production was not likely a factor contributing to follicular size or reduced pregnancy rate to AI.

342 CANADIAN JOURNAL OF ANIMAL SCIENCE Effects of day 14 Post-AI GnRH In contrast to studies in dairy cattle (Rettmer et al. 1992; Drew and Peters 1994), injection of GnRH during the midluteal phase after AI did not improve pregnancy rates in suckled beef cows. As with synchronization, midluteal phase treatment with GnRH was expected to cause release of luteinizing hormone, which was expected to enhance progesterone production by the corpus luteum and cause large follicles to ovulate or luteinize, thereby decreasing plasma estrogen concentrations (Mann and Lamming 1995). In nonpregnant cattle, estrogen produced by a large ovarian follicle (approximately 15 d after estrus) enhances release of prostaglandin F 2α from the uterus, causing the corpus luteum to regress. In pregnant cattle, a signal from the embryo (probably interferon-tau) suppresses prostaglandin F 2α production and maintains the corpus luteum and pregnancy (Thatcher et al. 1997). However, only 54% of cows ovulate in response to GnRH given in mid-luteal phase (Vasconceles et al. 1999) and the ovulatory capacity of the dominant follicle at this time may be dependent upon whether cows have two or three waves of folliclular growth. Treatment with human chorionic gonadotropin hormone 7 d after AI (Rajamahendran and Sianangama 1992) improved pregnancy rates in dairy cows. Post-AI treatment with GnRH is likely to be more effective on day 7 than day 14 because 96% of cows ovulate in response to GnRH given in the early (day 5 to day 9) luteal phase (Vasconceles et al. 1999). Others (Mee et al. 1993; Twagiramungu et al. 1995a) have reported that GnRH given at estrus or 7 d later resulted in increased plasma progesterone and corpora lutea with a greater number of large luteal cells. This may explain, in part, why apparent embryonic mortality was much less with than without GnRH after. SUMMARY AND CONCLUSIONS This study examined the effect of GnRH in -based timed-insemination programs for crossbred beef cows at different stages of lactation in spring and fall. Double injection of (DPG) was compared to Ovsynch (OVS) and modified Ovsynch (BRC) treatments, with and without 14 d post-ai GnRH treatment. The use of GnRH in the synchronization program improved timed-ai pregnancy rates 57% and resulted in a more compact calving season. Injection of GnRH 14 d after AI had no effect on pregnancy, conception or day 21 progesterone concentration. There was no advantage to administering the second GnRH 18 h before (OVS) rather than at the time (BRC) of AI (66 h after ). Therefore, the three-step BRC method (initial injection of GnRH, 7 d later, GnRH and AI 66 h after ) has the potential to be a suitable timed-ai program for commercial beef cows. ACKNOWLEDGEMENTS Authors thank D. Sykes (Senior Herdsperson), R. Kristjansson (Junior Herdsperson) and all Beef Program staff for their assistance in conducting this research. D. Lischka (ABS Canada, St Jacobs) and C. Ross (ABS Canada Ltd., Western Region) are thanked for their technical assistance with semen handling and AI. This research was funded by the Agriculture and Agri-Food Matching Investment Initiative and Ayerst Veterinary Laboratories (Factrel ), Pharmacia & Upjohn (Lutalyse ) and American Breeder Service (ABS) Canada (semen). Agriculture Canada 1990. Recommended code of practice for the care and handling of farm animals: Beef Cattle. Agriculture Canada. Ottawa. Publ. No. 1870/E. Berardinelli, J. G. and Adair, R. 1989. Effect of prostaglandin F2 α dosage and stage of estrous cycle on the estrous response and corpus luteum function in beef heifers. Theriogenology 32: 301 314. Canadian Council on Animal Care. 1993. Guide to the care and use of experimental animals. Volume 1. 2nd ed. CCAC, Ottawa, ON. Crowe, M. A., Goulding, D., Baguisi, A., Boland, M. P. and Roche, J. F. 1993. Induced ovulation of the first postpartum dominant follicle in beef suckler cows using a GnRH analogue. J. Reprod. Fertil. 99: 551 555. Del Vecchio, R. P., Sutherland, W. D. and Connor, L. M. 1995. A solid phase enzyme-immunoassay for the determination of progesterone in bovine, porcine and ovine plasma. Can. J. Anim. Sci. 75: 525 529. Drew, S. B. and Peters, A. R. 1994. Effect of buserelin on pregnancy rates in dairy cows. Vet. Rec. 134: 267 269. Geary, T. W. and Whittier, J. C. 1998. Effect of a timed insemination following synchronization of ovulation using the Ovsynch or CO-synch protocol in beef cows. Prof. Anim. Sci. 14: 217 220. Geary, T. W., Whittier, J. C., Downing, E. R., LeFever, D. G., Silcox, R. W., Holland, M. D., Nett, T. M. and Niswender, G. D. 1998. Pregnancy rates of postpartum beef cows that were synchronized with the Synchro-Mate B or Ovsynch protocol. J. Anim. Sci. 76: 1523 1527. Ginther, O. J., Knopf, L. and Kastelic, J. P. 1989. Temporal associations among ovarian events during estrous cycles with two or three waves. J. Reprod. Fertil. 87: 223 230. Hafez, E. S. E. 1987. Transport and survival of gametes. Page 187 in E. S. E. Hafez, ed. Reproduction in farm animals. 5th ed. Lea & Febiger, Philadelphia, PA. Kastelic, J. P., Curran, S., Pierson, R. A. and Ginther, O. J. 1988. Ultrasonic evaluation of the bovine conceptus. Theriogenology 29: 39 54. Kastelic, J. P., Knopf, L. and Ginther, O. J. 1991a. Effect of day of prostaglandin F2( treatment on selection and development of the ovulatory follicle in heifers. Anim. Reprod. Sci. 23: 169 180. Kastelic, J. P., Northey, D. L. and Ginther, O. J. 1991b. Spontaneous embryonic death on days 20 to 40 in heifers. Theriogenology 35: 351 363. King, M. E., Kiracofe, G. H., Stevenson, J. S. and Schalles, R. R. 1982. Effect of stage of the estrous cycle on interval to estrus after PGF2( in beef cattle. Theriogenology 18: 191 200. Macmillan, K. L. and Thatcher, W. W. 1991. Effects of an agonist of gonadotropin-releasing hormone on ovarian follicles in cattle. Biol. Reprod. 45: 883 889. Mann, G. E. and Lamming, G. E. 1995. Effects of treatment with buserelin on plasma concentrations of oestradiol and progesterone and cycle length of the cow. Br. Vet. J. 51: 427 432. Mee, M. O., Stevenson, J. S., Scolby, R. K. and Folman, Y. 1993. Influence of gonadotropin-releasing hormone and timing of insemination relative to estrus on pregnancy rates of dairy cattle at first service. J. Dairy Sci. 73: 1500 1507. Milvae, R. A., Murphy, B. D. and Hansel, W. 1984. Prolongation of the bovine estrous cycle with a gonadotropinreleasing hormone analogue. Biol. Reprod. 31: 664 670.

Odde, K. G. 1990. A review of synchronization of estrus in postpartum cattle. J. Anim. Sci. 68: 817 830. Peters, K. E., Bergfeld, E. G., Cupp, A. S., Kojima, F. N., Mariscal, V., Sanchez, T., Wherman, M. E., Grotjan, H. E., Hamernik, D. L. and Kittok, R. J. 1994. Luteinizing hormone has a role in development of fully functional corpora lutea (CL) but is not required to maintain CL function in heifers. Biol. Reprod. 51: 1248 1254. Pursley, J. R., Mee, M. O. and Wiltbank, M. C. 1995. Synchronization of ovulation in dairy cows using PGF2 alpha and GnRH. Theriogenology 44: 915 923. Pursley, J. R., Kosorok, M. R. and Wiltbank, M. C. 1997. Reproductive management of lactating dairy cows using synchronization of ovulation. J. Dairy Sci. 80: 301 306. Pursley, J. R., Silcox, R. W. and Wiltbank, M. C. 1998. Effect of time of artificial insemination on pregnancy rates, calving rates, pregnancy loss, and gender ratio after synchronization of ovulation in lactating dairy cows. J. Dairy Sci. 81: 2139 2144. Rajamahendran, R. and Sianangama, P. C. 1992. Effect of human chorionic gonadotrophin on dominant follicles in cows: formation of accessory corpora lutea, progesterone production and pregnancy rates. J. Reprod. Fertil. 95: 577 584. Rettmer, I., Stevenson, J. S. and Corah, L. R. 1992. Pregnancy rates in beef cattle after administering a GnRH agonist 11 to 14 days after insemination. J. Anim. Sci. 70: 7 12. Richards, M. W., Spitzer, J. C. and Warner, M. B. 1986. Effect of varying levels of postpartum nutrition and body condition at calving on subsequent reproductive performance in beef cattle. J. Anim. Sci. 62: 300 306. Rorie, R. W., Lester, T. D., Lindsey, B. R. and McNew, R. W. 1999. Effect of timing artificial insemination on gender ratio in beef cattle. Theriogenology 52: 1035 1041. Roy, G. L. and Twagiramungu, H. 1997. Relationship between onset of estrus, time of GnRH administration and time of AI after prostaglandin-induced luteolysis in cattle. Theriogenology 47: 150 (Abstr.). Savio, J. D., Thatcher, W. W., Morris, G. R., Entwistle, K., Drost, M. and Mattiacci, M. R. 1993. Effects of induction of low plasma progesterone concentrations with a progesterone-releasing intravaginal device on follicular turnover and fertility in cattle. J. Reprod. Fertil. 98: 77 84 SMALL ET AL. TIMED-AI OF BEEF COWS 343 SAS Institute, Inc. 1988. SAS/STAT user s guide (Release 6.03). SAS Institute, Inc., Carry, NC. Small, J. A. and McCaughey, W. P. 1999. Beef cattle management in Manitoba. Can. J. Anim. Sci. 79: 539 544 Stevenson, J. S. 2000. Use of GnRH to synchronize estrus and (or) ovulation in beef cows with or without timed insemination. Pages 37 45 in 49th Annual Beef Cattle Short Course Proceedings, Biotechnologies of Reproductive Biology 3 5 May, University of Florida, Gainesville, FL. Tanabe, T. Y., Deaver, D. R. and Hawk, H. W. 1994. Effect of gonadotropin-releasing hormone on estrus, ovulation and ovum cleavage rates of dairy cows. J. Anim. Sci. 72: 719 724 Thatcher W. W., Binelli, M., Burke, J., Staples, C. R., Ambrose, J. D. and Coelho, S. 1997. Antiluteolytic signals between the conceptus and endometrium. Theriogenology 47: 131 140 Twagiramungu, H., Guilbault, L. A., Proulx, J. G. and Dufour, J. J. 1995a. Buserelin alters the development of the corpora lutea in cyclic and early postpartum cows. J. Anim. Sci. 73: 805 811. Twagiramungu, H., Guilbault, L. A., Proulx, J., Villeneuve, P. and Dufour, J. J. 1994. Influence of corpus luteum and induced ovulation on ovarian follicular dynamics in postpartum cyclic cows treated with buserelin and cloprostenol. J. Anim. Sci. 72: 1796 1805. Twagiramungu, H., Roy, G. L., Laverdi(re, G. and Dufour, J. J. 1995b. Fixed-time insemination in cattle after synchronization of estrus and ovulation with GnRH and prostaglandin. Theriogenology 43: 341 (Abstr). Vasconceles, J. L. M., Silcox, R. W., Rosa, G. J. M., Pursley, J. R. and Wiltbank, M. C. 1999. Synchronization rate, size of the ovulatory follicle, and pregnancy rate after synchronization of ovulation beginning on different days of the estrous cycle in lactating dairy cows. Theriogenology 52: 1067 1078. Wehner, G. R., Wood, C., Tague, A., Barker, D. and Hubert, H. 1997. Efficiency of the OVATEC unit for estrus detection and calf sex control in beef cows. Anim. Reprod. Sci. 46: 27 34. Wiltbank, J. N. 1970. Research needs in beef cattle reproduction. J. Anim. Sci. 31: 755 762.