Use of Intravaginal Progesterone-Releasing Device (CIDR 1 ) for Timed Artificial Insemination (AI) in Crossbred Cattle of Bos indicus Breeding 2,3

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1 Use of Intravaginal Progesterone-Releasing Device (CIDR 1 ) for Timed Artificial Insemination (AI) in Crossbred Cattle of Bos indicus Breeding 2,3 Janis Fullenwider Ray Kempfer Christin Barthle Willard Lemaster Joel Yelich 4 Utilization of the CIDR for estrous synchronization followed by timed-ai in crossbred cattle of Bos indicus breeding has potential to yield acceptable AI pregnancy rates (40-60%) in both lactating and non-lactating cows. Summary Two estrous synchronization and timed-ai protocols were compared in crossbred cattle of Bos indicus breeding. In experiment 1, 548 dry cows received a 7-d CIDR with 2 ml of estradiol benzoate at CIDR insertion and 5 ml of prostaglandin F2a (Lutalyse) at CIDR removal. Treatment 1 received 1 ml of estradiol benzoate 24 h after CIDR removal while treatment 2 received 2 ml of GnRH (Cystorelin) at AI. Both treatments were AI approximately h after CIDR removal. Experiment 2 utilized 390 lactating, postpartum crossbred Bos indicus cows. The same treatments used in experiment 1 were used for experiment 2. Calves were removed from cows at CIDR removal and returned after the cows were inseminated in experiment 2. In experiment 1, AI pregnancy rates were similar between the estradiol benzoate treatment (47.2%) and GnRH treatment (46.6%); while in experiment 2, AI pregnancy rates were greater (P <.05) for the estradiol benzoate (53.3 %) than GnRH treated cows (43.5 %). The number of cows becoming pregnant in the first 30 d of the breeding season was > 75% across all treatments and experiments. In both experiments, AI pregnancy rates for the semen used from 20 different sires used ranged from 0-80%. In conclusion, both CIDR estrous synchronization protocols were effective timed-ai protocols resulting in acceptable AI pregnancy rates. However, overall effectiveness of timed-ai protocols may be limited by the fertility of the frozen semen used. Introduction The majority of beef cattle in Florida contain a percentage of Bos indicus breeding. However, synchronization of estrus in cattle of Bos indicus breeding is difficult for several reasons. First, estrus is difficult to detect due to its short duration and it usually occurs during the late evening or early morning hours. Second, cattle of Bos indicus breeding respond differently to many of the estrous synchronization products used today than cattle of Bos taurus breeding. Estrus detection can be a time and labor consuming process, which makes artificial insemination (AI) programs impractical for some beef cattle producers. Estrus detection can be eliminated by breeding animals at a designated time, also known as timed-ai or mass mating. However, pregnancy rates are generally decreased in timed-ai programs compared to breeding cattle by behavioral estrus. During the past few years at the University of Florida, we have been conducting estrous synchronization research using an intravaginal progesterone-releasing device known as the EAZI-BREED CIDR which is produced in New Zealand. The CIDR is a modified plastic device containing natural progesterone that is inserted into the vagina of the cow for 7 d and prevents the cow from exhibiting estrus. However, using progestagen based treatments for extended periods (>7 d) can result in the maintenance of a large ovarian follicle by the end of the progestagen treatment. Fertility of this large and persistent follicle is decreased when it is ovulated after withdrawal of the progestagen. Recent research conducted in our laboratory reported that estrogen administered when the CIDR was inserted, initiated a premature death of the largest follicle on the ovary, resulting in recruitment and development of a new and fertile follicle that ovulates after the CIDR is removed.

2 Therefore, the objectives of this research were to determine if estrogen administered at CIDR insertion increased pregnancy rates compared with no estrogen and to evaluate pregnancy rates of cows inseminated either by observed estrus or timed-ai in a large field trial. Procedure The research project was conducted in two experiments. In experiment 1, 548 crossbred, dry cows sired by either Red Brangus, Simbrah or Braford sires were used. The experiment was replicated at four locations on the same ranch. On D 0 of the experiment, cows were assigned a body condition score using a 1 to 9 scale (1 = very thin; 5 = moderate; 9=very fat) and were allotted randomly to treatment within each respective location. All cows received a 2 ml injection of estradiol benzoate (1 mg/ml; CIDROL, InterAg, Hamilton, NZ) at CIDR (EAZI-BREED CIDR, InterAg, Hamilton, NZ) insertion (D 0) and 5 ml of prostaglandin F2a (Lutalyse; Pharmacia & Upjohn, Kalamazoo, MI) on D 7 of the experiment. Treatment 1 received a 1 ml injection of estradiol benzoate (1 mg/ml) 24 h after CIDR removal while treatment 2 received a 2 ml injection of GnRH (100 mg Cystorelin; Merial Limited Athens, GA) at insemination. Both treatment groups were inseminated approximately h after CIDR removal. Semen from 10 different sires was allotted equally to each treatment within a location. In experiment 2,390 crossbred, lactating and postpartum Bos indicus cows were synchronized with the same treatments used in experiment 1. Cows were managed in two age groups and were either 3 y of age (n =127; 3/4 Red Angus - 1/8 Brahman - 1/8 English) or > 4 years of age (n = 263; 1/2 Red Angus - 1/4 Brahman - 1/4 English). On D 0 of the experiment, cows were assigned a body condition score and were allotted randomly to treatment within each respective age group. Calves were separated from cows starting at CIDR removal and until AI was completed at which time they were placed back with the cows. Semen from 10 different sires was allotted equally to each treatment within an age group and was assigned randomly to cows as they were inseminated. In both experiments bulls were turned in with cows approximately d after AI and pregnancy was determined at approximately d after AI using ultrasonography. Results Approximately 47% of the cows became pregnant to the timed-ai in experiment 1 and there were no significant differences in AI pregnancy rates between treatments and locations (Table 1) or between cow sire (Table 2). Our ultimate goal is to develop a timed-ai protocol that will consistently yield AI pregnancy rates between 55-60%. However, numerous factors can influence the effectiveness of any estrous synchronization system. In experiment 1, there was a significant effect of sire on pregnancy rates. s for individual sires ranged from 25% to 61.4% (Table 2). There was no treatment by sire interaction, indicating that pregnancy rates for individual sires were similar across the two treatments. Although all animals in this experiment were inseminated over a 4-h period, there is a larger range of time over which the animals are probably ovulating, approximately h after their respective treatments. Therefore, one explanation for the large range in fertility between sires could be the sperms ability or inability to survive in the reproductive tract over this extended period of time. Therefore, differences in fertility between sires could be a major factor limiting the effectiveness of timed-ai programs. In experiment 2, the estradiol benzoate treated (53.3 %) cows had greater (P <.05) AI-pregnancy rates than the GnRH treated (43.5 %) cows. The magnitude of difference was significant in the cows that were > 4 y of age but not in cows that were 3 y of age. Since the body condition score of the 3-y-old cows (BCS = 4.3) was considerably less than the older cows (BCS = 5.0), it can be postulated that fewer of the 3-y-old cows were cycling when the CIDR was inserted. As with experiment 1, there was a significant effect of the sire on AI-pregnancy rates. However, the sire effect appears to be less of a factor in the estradiol benzoate treated cows than GnRH treated cows (Table 4). One theory could be that in the estradiol benzoate treated cows, ovulation is induced earlier (54-60 h after CIDR removal) compared to ovulation induced with GnRH treatment ( 72 h after CIDR removal). Therefore, AI is more closely timed with ovulation in the estradiol benzoate treated cows than GnRH treated cows. Pregnancy data from individual sires supports this theory. Sires that exhibited "low fertility" in the GnRH treated cows were of "normal to high fertility" in EB-treated cows (Table 4). This data suggests that semen from "high fertility" sires may survive longer in the reproductive tract than semen from "low fertility" sires. Consequently, low fertility sires may be compensated for in the estradiol benzoate treated cows due to the shorter length of time between insemination and ovulation. However, additional research must be conducted to determine if this theory is correct. When 30-d pregnancy rates were compared across treatments for both experiments, greater than 75 % of the cows became pregnant in the first 30 d of the breeding season. As a result, over two-thirds of the calves are going to be born early during the subsequent calving season resulting in a more uniform and heavier calf crop at weaning; an often-overlooked benefit of estrous synchronization.

3 In conclusion, both CIDR estrous synchronization systems appear to be effective timed-ai protocols in both lactating and dry crossbred cows of Bos indicus breeding. However, maximum effectiveness of timed-ai protocols may be limited due to differences in fertility of semen. Although, the GnRH protocol would probably be favored by producers since cattle are handled only 3 times versus 4 times with the estradiol benzoate treatment. Additional research is currently being conducted with the CIDR in an attempt to increase timed-ai pregnancy rates. 1 The CIDR has yet to be approved for use as an estrous synchronization agent by the Food and Drug Administration (FDA) in the United States. 2 The aut hors would like to th ank all of the staff at Deseret Cattle and Citrus, St. Cloud, FL and Broseco Ranch, Omaha, TX for their assistance and use of cattle to conduct these experiments. The authors would also like to thank all of the University of Florida undergraduates who assisted with this experiment. 3 The authors would also like to thank InterAg, Hamilton New Zealand for donation of the CIDR and estradiol benzoate; Pharmacia Upjohn for donation of the Lutalyse; and Merial Limited for donation of the Cystorelin and the American Simmental Association for financial support to conduct this research. 4 Janis Fullenwider, graduate student; Ray Kempfer, former graduate student; Christin Barthle, bilogical scientist; Department of Animal Sciences, Gainesville; Williard Lemaster, former Livestock Programs Coordinator; Joel Yelich, Assistant Professor, Department of Animal Sciences, Gainesville. Table 1. s by treatment and location for crossbred cows of Bos indicus breeding synchronized with a controlled intravaginal progesterone-releasing device (CIDR), and timed-ai 48 h (TAI) after CIDR removal Variable Treatments a N (%) b 30 Day EB 24 h post CIDR + TAI TAI/GnRH Locations d 1 (BCS 4.9) (BCS 4.8) (BCS 4.1) (BCS 4.3) a Start of the experiments designated as day 0. All treatments received a 2 ml injection of estradiol benzoate (EB) at CIDR insertion and 5 ml prostaglandin F 2 (Lutalyse) on day 7. Treatment 1 received 1 ml EB 24 hours after CIDR removal. Insemination treatments were: 1) TAI 48 hours after CIDR removal; 2) TAI 48 hours after CIDR removal with 2 ml Cystorelin (GnRH) at TAI. b Percentage of animals becoming pregnant to TAI of the total treated. c Percentage of animals becoming pregnant during the first 30-d of the breeding season of the total treated. d BCS=body condition score (1 = emaciated; 5 = moderate; 9 = very fat)

4 Table 2. s by treatment and location for crossbred cows of Bos indicus breeding synchronized with a controlled intravaginal progesterone-releasing device (CIDR), and timed-ai 48 h (TAI) after CIDR removal a Variable N (%) b 30-d Sires of cows AI Sires (P<.001) Simbrah Brangus Braford A B C D E F G H I J a Start of the experiments designated as D 0. All treatments received a 2 ml injection of estradiol benzoate (EB) at CIDR insertion and 5 ml prostaglandin F 2 (Lutalyse) on D 7. Treatment 1 received 1 ml EB 24 h after CIDR removal. Insemination treatments were: 1) TAI 48 h after CIDR removal; 2) TAI 48 h after CIDR removal with 2 ml Cystorelin (GnRH) at TAI. b Percentage of animals becoming pregnant to TAI of the total treated. c Percentage of animals becoming pregnant during the first 30-d of the breeding season of the total treated.

5 Table 3. s by treatment and age group for lactating, crossbred cattle of Bos indicus breeding synchronized with a controlled intravaginal progesterone-releasing device (CIDR) and timed-ai 48 h (TAI) after CIDR Variable N Pregnancy rates (%) b 30-d s Treatments a 1) EB 24 h post CIDR + TAI * ) TAI/GnRH Age group (P<.01) d Cow age 4 (BCS = 5.0) 1) EB 24 h post CIDR + TAI * ) TAI/GnRH Cow age <3 (BCS = 4.3) 1) EB 24 h post CIDR + TAI ) TAI/GnRH a Start of the experiment designated as D 0. All treatments received a 2 ml injection of estradiol benzoate (EB) at CIDR insertion and 5 ml prostaglandin F2 (Lutalyse) on D 7. Treatment 1 received 1 ml EB 24 h after CIDR removal. Insemination treatments were: 1) TAI 48 h after CIDR removal; 2) TAI 48 h after CIDR removal with 2 ml Cystorelin (GnRH) at TAI. b Percentage of animals becoming pregnant to TAI of the total treated. c Percentage of animals becoming pregnant during the first 30-d of the breeding season of the total treated. d BCS=body condition score (1 = emaciated; 5 = moderate; 9 = very fat) Table 4. s by AI sire for lactating, crossbred cows of Bos indicus breeding synchronized with a controlled intravaginal progesterone-releasing device (CIDR) and timed-ai 48 h (TAI) after CIDR removal a Variable AI sires (P <.01) Treatment 1 (EB) s (%) b Treatment 2 (GnRH) s (%) b K 14/19 = /20 = 50.0 L 19/35 = /34 = 61.8 M 6/10 = /10 = 60.0 N 7/10 = /10 = 0.0 O 11/21 = /20 = 20.0 P 14/35 = /35 = 62.9 Q 16/20 = /20 = 35.0 R 11/21 = /21 = 33.3 S 4/13 = /12 = 41.7 T 4/9 = /9 = 22.2