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J. Anim. Sci. 2002. 80:3060-3064
© 2002 American Society of Animal Science

Evaluation of a fixed-time artificial insemination protocol for postpartum suckled beef cows1

G. A. Perry, M. F. Smith and D. J. Patterson2

Department of Animal Science, University of Missouri, Columbia 65211

2 Correspondence:
S132 Animal Science Research Center (phone: 573-882-7519; fax: 573-884-4545; E-mail:
PattersonD{at}missouri.edu).


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
Treatment with melengestrol acetate (MGA), an oral progestin, prior to administration of gonadotropin-releasing hormone (GnRH) and prostaglandin F2{alpha} (PG) effectively synchronizes estrus and maintains high fertility in postpartum beef cows. The objective of this experiment was to determine whether treatment with MGA prior to a GnRH-PG-GnRH protocol would improve pregnancy rates resulting from fixed-time artificial insemination (AI). Multiparous crossbred beef cows at two University of Missouri-Columbia farms (n = 90 and n = 137) were assigned by age and days postpartum to one of two treatments. Cows were fed carrier (1.8 kg•animal-1•d-1) with or without MGA (0.278 mg•kg-1) for 14 d. All cows were administered GnRH (100 µg; intramuscularly) on d 12 after MGA or carrier withdrawal and 7 d before PG (25 mg; intramuscularly). All cows received a second injection of GnRH and AI 72 h after PG. Mean days postpartum for MGA and control cows at the initiation of treatment were 39.6 and 38.9 d for herd 1; and 51.9 and 50.9 d for herd 2, respectively (P > 0.70 within herds). Blood samples were collected from all cows at 10 and 1 d before the feeding of MGA or carrier began and at the times GnRH and PG were administered. Concentrations of progesterone in serum at the initiation of treatment were elevated (>1 ng/mL) in 0% of MGA and 7% of control cows in herd 1, and 54% of MGA and 49% of control cows in herd 2 (P > 0.05 within herds). Pregnancy rates to fixed-time AI were determined by transrectal ultrasonography 50 d after AI. Pregnancy rates in herd 1 were 58% (26/45) and 51% (23/45) for MGA-treated and control cows, respectively (P = 0.52), and 63% (44/70) and 45% (30/67) for MGA-treated and control cows in herd 2, respectively (P = 0.03). Differences in pregnancy rates to fixed-time AI were significant (P = 0.04) when data from the two herds were combined (with MGA = 70/115 [61%]; control = 53/112 [47%]). There was no difference (P > 0.20) in final pregnancy rates (timed AI plus 45 d exposure to bulls) between treatments, within herds, or when herds were combined. In summary, pregnancy rates resulting from fixed-time AI may be improved with treatment of MGA prior to a GnRH-PG-GnRH protocol.

Key Words: Artificial Insemination • Beef Cows • Estrus • Synchronization


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
The development of a successful fixed-time AI protocol for beef cows will require precise control of the estrous cycle by coordinating both follicular growth and luteal regression. Melengestrol acetate (MGA) is an orally active progestin that is effective for synchronizing estrus in replacement beef heifers (Brown et al., 1988) and postpartum suckled beef cows (Patterson et al., 1995). When anestrous postpartum beef cows were fed MGA for 14 d, up to 20% of the cows exhibited a short cycle and did not have luteal tissue capable of responding to PG on d 17 after MGA withdrawal (Fralix et al., 1996). Injection of a GnRH agonist can effectively ovulate a large dominant follicle and form a new corpus luteum (CL). In addition, a new follicular wave will emerge within 3 to 4 d (Twagiramungu et al., 1995). Administration of GnRH 7 d prior to PG offers the potential either to reduce the incidence or eliminate the occurrence of short cycles. When GnRH was added to the MGA-PG (14/17) protocol on d 10 after MGA withdrawal, synchrony of estrus was improved compared to that of cows that did not receive GnRH. Over 80% of GnRH-treated cows exhibited estrus 48 to 96 h after PG (Patterson et al., 2000).

In heifers, administering PG 19 d after MGA withdrawal resulted in improved synchrony of estrus and higher pregnancy rates compared with heifers administered PG on d 17 (Lamb et al., 2000). Wood et al. (2001) reported improved synchrony of estrus and resulting ovulation using a modified MGA-PG protocol that called for the addition of GnRH on d 26 of treatment (MGA feeding d 1 to 14; GnRH on d 26; PG on d 33). More recently, Patterson et al. (2001) characterized estrus response and resulting fertility in postpartum suckled beef cows that were synchronized using this modified protocol. Based on these studies, the objective of this experiment was to determine whether pretreatment with MGA prior to a GnRH-PG-GnRH protocol would improve pregnancy rates resulting from fixed-time AI.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Implications
 Literature Cited
 
Experimental Design

This experiment was conducted with two herds from the University of Missouri-Columbia system. Postpartum suckled beef cows were assigned to one of two treatments (MGA or control) based on calving date or days postpartum, and cow age. Control and MGA-treated cows received supplement (1.8 kg•animal-1•d-1 of Cattle Charge; MFA, Columbia, MO) with or without MGA (0.278 mg•kg-1) for 14 d. On d 26 of treatment (12 d after withdrawal of carrier with or without MGA), all cows were injected with GnRH (100 µg as 2 mL of Cystorelin i.m.; Merial, Athens, GA). Seven days later (d 33 of treatment), all cows were injected with PG (25 mg as 5 mL of Lutalyse i.m.; Pharmacia Animal Health, Kalamazoo, MI). Seventy-two hours after PG (72 h was the peak estrus response following the MGA-GnRH-PG protocol as characterized by Patterson et al., 2001) all cows received a second injection of GnRH (Cystorelin; 100 µg) and were artificially inseminated (Figure 1Go). Within each herd, cows were assigned equally to one of two inseminators by treatment, age, and days postpartum, and all cows in each herd were inseminated with semen from the same bull.



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  		Figure 1. Treatment schedules for MGA-GnRH-PG-GnRH (MGA) and GnRH-PG-GnRH (control) protocols. MGA = melengestrol acetate, PG = prostaglandin F2{alpha}, GnRH = gonadotropin-releasing hormone.

 
Control and MGA-treated cows at the time treatments were initiated in herd 1 were 40 ± 2.1 and 39 ± 2.1 d postpartum, respectively. Cows in herd 2 were 51 ± 2.0 d and 52 ± 2.0 d postpartum for control and MGA groups, respectively (Table 1Go). All cows were maintained in treatment groups on pasture and allowed to graze throughout the entire project. Carrier with or without MGA was fed as a supplement to grazing. Cow BCS (1 to 9; 1 = emaciated and 9 = obese) was determined at AI. Bulls were exposed to cows beginning 15 d after AI for 45 d, and pregnancy rates to fixed-timed AI were determined by transrectal ultrasonography 50 d following insemination. Final pregnancy rate was determined 45 d after the end of the breeding period.


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  		Table 1. Number of cows at each location, days postpartum, body condition score (BCS), and estrous-cycling status prior to initiation of treatment
 
Blood Collection and Radioimmunoassays

Blood samples for progesterone were collected by jugular venipucture into 10-mL Vacutainer tubes (Fisher Scientific, Pittsburgh, PA) 10 d and 1 d before treatment began, and at the time GnRH (d 26) and PG (d 33) were administered. Cows with serum concentrations of progesterone >=1 ng/mL at either one or both of the pretreatment sampling times were considered to be estrous-cycling. Blood was allowed to clot for 1 h at room temperature, stored at 4°C for 24 h, and centrifuged at 2,000 x g for 30 min to harvest serum. Serum was stored at -20°C until assayed for progesterone. Serum concentrations of progesterone were analyzed by RIA (Kirby et al., 1997; Diagnostic Products Corporation, Los Angeles, CA). Intra- and interassay CV for progesterone assays were 2.2% and 6.9%, respectively, and assay sensitivity was 0.5 ng/mL of serum.

Statistical Analysis

Differences in days postpartum and BCS between treatments were analyzed by ANOVA in SAS (SAS Inst. Inc., Cary, NC). The preceding variables were analyzed for an effect of treatment, herd, and treatment x herd interaction. Mean separation was performed using LSD (means ± SEM; Snedecor and Cochran, 1989) when the F statistic was significant (P < 0.05). Differences between treatments in estrous-cycling status, presence of luteal tissue, conception rates (number of animals pregnant to timed AI/total number of animals), final pregnancy rates (number of animals pregnant at the end of the breeding season/total number of animals), and inseminator conception rates were analyzed by chi-squared analysis using the frequency procedure of SAS (Snedecor and Cochran, 1989).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Implications
 Literature Cited
 
The number of cows per treatment, days postpartum, BCS, and estrous-cycling status for cows in both herds are shown in Table 1Go. Days postpartum did not differ (P > 0.70) between treatments within herd or when data from the two herds were combined (control, 47 ± 1.5 d; MGA, 47 ± 1.5 d). Body condition score was different (P < 0.01) between treatments in herd 1 (control, 5.7 ± 0.1; MGA, 5.3 ± 0.01), with control cows having a higher BCS than MGA-treated cows. There was no difference (P = 0.35) between treatments, however, in cow BCS (control, 5.7 ± 0.1; MGA, 5.6 ± 0.01) in herd 2. There was a treatment x location interaction for BCS; therefore BCS was not analyzed when data from both herds were combined. Percent of cows estrous-cycling before the initiation of treatment was not different in herd 1 (P = 0.07; 0 vs 7%) or in herd 2 (P = 0.55; 49 vs 54%) for control and MGA-treated cows, respectively. In addition, there was no difference (P = 0.88) in the percent of cows estrous-cycling before the start of treatment when data from the two herds were combined (control, 32%; MGA, 33%). The percentage of cows that were estrous-cycling at the initiation of treatment between herds, however, was different (P < 0.01).

The percentage of cows with luteal tissue (progesterone concentrations >=1 ng/mL) at the first GnRH injection and at PG administration are shown in Table 2Go. At the time of the first GnRH injection in herd 1, 60% of the control cows and 67% of the MGA-treated cows had elevated concentrations of progesterone in serum (P = 0.51). More cows fed MGA in herd 2 (P = 0.01), and when data from the two herds were combined (P = 0.02) (herd 2, 82%; combined, 77%), had elevated concentrations of progesterone in serum at GnRH compared to controls in herd 2 (64%) or when data from the two herds were combined (63%). There was no difference in the percentage of cows with elevated (>1 ng/mL) concentrations of progesterone in serum at the time PG was administered to either herd (herd 1, P = 0.82; herd 2, P = 0.26), or when data from the two herds were combined (P = 0.50).


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  		Table 2. Presence of luteal tissue based on concentrations of progesterone (P4) in serum obtained at the first gonadotropin-releasing hormone (GnRH) injection and at prostaglandin F2{alpha} (PG) for cows assigned to the MGA or control treatments
 
Conception rates in herd 1 resulting from fixed-time AI (control, 51%; MGA, 58%) did not differ (P = 0.52) between treatments. Conception rates resulting from fixed-time AI, however, were different (P = 0.03) between treatments in herd 2 (control, 45%; MGA, 63%), with higher pregnancy rates found among MGA-treated cows. When data from both herds were combined (control, 47%; MGA, 61%; Table 3Go), a higher proportion (P = 0.04) of cows that received MGA prior to the GnRH-PG-GnRH protocol became pregnant after fixed-time AI. There was no difference (P = 0.21) in conception rate between inseminators in either herd. Final pregnancy rates did not differ between treatments in either herd (herd 1 control, 84%; MGA, 84%; P = 1.0; herd 2 control, 88%; MGA, 91%; P = 0.51) or when pregnancy rates from the two herds were combined (control, 87%; MGA, 89%; P = 0.63; Table 4Go).


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  		Table 3. Conception rate after fixed-time artificial insemination among cows assigned to the MGA or control treatments
 

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  		Table 4. Final pregnancy rate among cows assigned to the MGA or control treatments
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Implications
 Literature Cited
 
Two methods of synchronizing estrus in anestrous and estrous-cycling beef cows include the GnRH-PG and MGA-PG protocols; however, these estrus-synchronization protocols have drawbacks. One limitation of the GnRH-PG method is that approximately 5 to 15% of the cows are detected in estrus on or before the day of PG injection, thus reducing the proportion of females that are detected in estrus and inseminated during the synchronized period (Twagiramungu et al., 1995; Kojima et al., 2000). A major disadvantage of the MGA-PG protocol is that up to 20% of anestrous cows exhibit a short luteal phase after the period of MGA treatment (Fralix et al., 1996), which in some cases necessitates a second PG injection.

The development of an effective fixed-time AI protocol, given these constraints, requires more precise control of follicular growth, CL regression, and ovulation. Various methods of synchronizing estrus prior to fixed-time AI have utilized GnRH to initiate a new follicular wave and control ovulation (Ov-Synch, Pursley et al., 1998; CO-Synch, Geary and Whittier, 1998). The protocol tested in this experiment involved a sequential approach, which combined treatment with MGA to inhibit ovulation (Zimbleman and Smith, 1966), GnRH to ovulate a dominant follicle and to initiate a new follicular wave (Twagiramungu et al., 1995), and PG to induce luteolysis. Cows were then inseminated 72 h after PG was administered, at which time a second injection of GnRH was administered.

At the time of the first GnRH injection, more MGA-treated cows in herd 2 had elevated concentrations of progesterone compared to cows fed the carrier without MGA. This supports previous studies that suggest that MGA successfully inhibits ovulation and synchronizes estrus and ovulation in cows so that they may be expected to exhibit estrus and ovulate 3 to 7 d after the last day of MGA (Patterson et al., 1989). Control cows could be expected to ovulate normally during the treatment period, with anestrous control cows able to spontaneously initiate estrous-cycling during the treatment period. Thus more MGA-treated cows would have luteal tissue present at the time GnRH was administered (d 26 of treatment) than control cows. In herd 1, there was no difference in the percentage of cows with elevated concentrations of progesterone at the time GnRH was administered. This is likely due to the lower number of cows that were estrous- cycling at the start of treatment in herd 1 compared with herd 2. Melengestrol acetate (0.5 mg•animals-1•d-1) will inhibit ovulation in estrous-cycling cows (Zimbleman and Smith, 1966), and following luteolysis, a persistent follicle will develop in response to low levels of the exogenous progestin (see review by Fortune and Rivera, 1999). Following the removal of MGA, estrous and ovulation usually occur within 3 to 7 d and increases the likelihood of luteal tissue being present in estrous-cycling cows on d 26. The administration of MGA (0.5 mg•animals-1•d-1) to anestrous postpartum beef cows, however, does not result in the formation of a persistent follicle and normal follicular waves continue (Perry et al., 2002). Thus, in herd 1, where very few cows were estrous- cycling at the initiation of treatment, the number of cows on d 26 of treatment with elevated concentrations of progesterone would not be expected to differ between treatments.

There was no difference in the number of cows with increased concentrations of progesterone at the time PG was administered in either herd. Since GnRH will induce ovulation of a dominant follicle (>10 mm; Twagiramungu et al., 1992; Ryan et al., 1998) and as it was administered 7 d preceding PG, cows with a dominant follicle at the time GnRH was administered would have had luteal tissue present at the time PG was given.

In herd 1, there was no difference (P = 0.52) between treatments in the percentage of cows that conceived to fixed-time AI. This could perhaps be due to the smaller total number of cows in this herd or the number of cows that were estrous-cycling before treatment began. Furthermore, there was no difference (P > 0.05) between treatments in the number of cows with elevated concentrations of progesterone at the first GnRH injection or at the time PG was administered. It is possible that pretreatment with MGA did not elicit an effect on cows in herd 1 since there were fewer cows estrous-cycling prior to treatment, and anestrous cows administered MGA experienced normal follicular turnover (Perry et al., 2002). In herd 2 (P = 0.03), and when data from both herds was combined (P = 0.04), a higher number of MGA-treated cows conceived to fixed-time AI compared to controls. These results support previous studies, which showed significant improvements in fertility among MGA-treated Holsteins at second service after MGA withdrawal (Britt et al., 1972), and among postpartum suckled beef cows that received MGA prior to PG administration (Patterson et al., 1995).

The higher pregnancy rate after fixed-time AI among MGA-treated cows is likely due to a percentage of cows not fed MGA exhibiting estrus and ovulating prior to PG administration. Hence, the potential number of cows among which precise control of estrus and ovulation occurred was likely reduced. Therefore, the pool of cows from which pregnancy was more likely to occur after fixed-time AI was larger among groups fed MGA.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
Literature Cited
 
Less than 5% of beef producers in the United States currently use estrus synchronization and artificial insemination in their herds. The reason producers do not utilize these procedures is largely attributed to the increased time and labor requirements associated with estrus detection. The development of a protocol that would enable producers to inseminate cows at a fixed time would eliminate the time and labor required to detect estrus. This protocol (MGA-GnRH-PG-GnRH) may offer significant potential to enhance results from fixed-time artificial insemination and may provide the opportunity to expand the use of artificial insemination in postpartum beef cows.


   Footnotes
 
1 Contribution from the Missouri Agriculture Experiment Station. This research was supported by Select Sires, Inc. and USDA-NRI 00-35203-9175. The authors gratefully acknowledge J. Bader for technical assistance; Merial for providing the Cystorelin, and Pharmacia Animal Health for providing the Lutalyse sterile solution for this research. Back

Received for publication April 16, 2002. Accepted for publication July 23, 2002.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 


Britt, J. H., E. Huertas Vega, and L. C. Ulberg. 1972. Managing reproduction in dairy cattle: 1. Progestogens for control of estrous in dairy cows. J. Dairy. Sci.55:598–605.[Abstract/Free Full Text]

Brown, L. N., K. G. Odde, M. E. King, D. G. LaFever, and C. J. Neubauer. 1988. Comparison of melengestrol acetate-prostaglandin F2{alpha} to synchro-mate B for estrous synchronization in beef heifers. Theriogenology 30:1–12.[Medline]

Fortune, J. E., and G. M. Rivera. 1999. Persistent dominant follicles in cattle: Basic and applied aspects. Arq. Fac. Vet. 27(Suppl.):24–36.

Fralix, K. D., D. J. Patterson, K. K. Schillo, R. E. Stewart, and K. D. Bullock. 1996. Change in morphology and corpora lutea, central luteal cavities and steroid secretion patterns of postpartum suckled beef cows after melengestrol acetate with or without prostaglandin F2{alpha}. Theriogenology 45:1255–1263.[Medline]

Geary, T. W., and J. C. Whittier.1998. Effects of a timed insemination following synchronization of ovulation using the Ovsynch or CO-Synch protocol in beef cows. Prof. Anim. Sci. 14:217–220.[Abstract/Free Full Text]

Kirby, C. J., M. F. Smith, D. H. Keisler, and M. C. Lucy. 1997. Follicular function in lactating dairy cows treated with sustained-release bovine somatotrophin. J. Dairy Sci. 80:273–285.[Abstract]

Kojima, F. N., B. E. Salfen, J. F. Bader, W. A. Ricke, M. C. Lucy, M. F. Smith, and D. J. Patterson. 2000. Development of an estrus synchronization protocol for beef cattle with short-term feeding of elengestrol acetate: 7-11 synch. J. Anim. Sci. 78:2186–2191.[Abstract/Free Full Text]

Lamb, G. C., D. W. Nix, J. S. Stevenson, and L. R. Corah. 2000. Prolonging the MGA-prostaglandin F2 alpha interval from 17 to 19 days in an estrous synchronization system for heifers. Theriogenology 53:691–698.[Medline]

Patterson, D. J., J. F. Bader, K. K. Graham, F. N. Kojima, G. A. Perry, M. S. Kerley, and M. F. Smith. 2001. Addition of GnRH to a melengestrol acetate (MGA)-prostaglandin F2{alpha} (PG) estrus synchronization protocol in postpartum beef cows. J. Anim. Sci. 79(Suppl. 1):1032 (Abstr.).[Abstract/Free Full Text]

Patterson, D. J., J. B. Hall, N. W. Bradley, K. K. Schillo, B. L. Woods, and J. M. Kearnan. 1995. Improved synchrony, conception rate, and fecundity in postpartum suckled beef cows fed melengestrol acetate prior to prostaglandin F2{alpha}. J. Anim. Sci. 73:954–959.[Abstract]

Patterson, D. J., G. H. Kiracofe, J. S. Stevenson, and L. R. Corah. 1989. Control of the bovine estrous cycle with melengestrol acetate (MGA): A review. J. Anim. Sci. 67:1895–1906.[Abstract/Free Full Text]

Patterson, D. J., S. L. Wood, F. N. Kojima, and M. F. Smith. 2000. Improved synchronization of estrus in postpartum suckled beef cows with a progestigin-GnRH- prostaglandin F2{alpha} (PG) protocol. J. Anim. Sci. Vol. 78(Suppl. 1):919 (Abstr.).[Abstract/Free Full Text]

Perry, G. A., F. N. Kojima, B. E. Salfen, J. F. Bader, D. J. Patterson, and M. F. Smith. 2002. Effect of an orally active progestin on follicular dynamics in cycling and anestrous postpartum beef cows. J. Anim. Sci. 80:1932–1938.[Abstract/Free Full Text]

Pursley, J. R., R. W. Silcox, and M. C. Wiltbank. 1998. Effect of time of artificial insemination on pregnancy rates, pregnancy loss, and gender ratio after synchronization of ovulation in lactating dairy cows. J. Dairy Sci. 81:2139–2144.[Abstract]

Ryan, M., M. Mihm, and J. F. Roche. 1998. Effect of GnRH given before or after dominance on gonadotrophin response and the fate of that follicle wave in postpartum dairy cows. J. Reprod. Fertil. Abst. Ser. 21:61.

Snedecor, G. W., and W. G. Cochran. 1989. Statistical Methods. 8th ed. Iowa State Univ. Press, Ames.

Twagiramungu, H., L. A. Guilbault, and J. J. Dufour. 1995. Synchronization of ovarian follicular waves with a gonadotropin-releasing hormone agonist to increase the precision of estrous in cattle: A review. J. Anim. Sci. 73:3141–3151.[Abstract]

Wood, S. L., M. C. Lucy, M. F. Smith, and D. J. Patterson. 2001. Improved synchrony of estrus and ovulation with the addition of GnRH to a melengestrol acetate— prostaglandin F2{alpha} synchronization treatment in beef heifers. J. Anim. Sci. 79:2210–2216.[Abstract/Free Full Text]

Zimbelman, R. G., and L. W. Smith. 1966. Control of ovulation in cattle with melengestrol acetate. II. Effects on follicular size and activity. J. Reprod. Fertil. 11:193–201.[Abstract/Free Full Text]


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