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Comparison of GnRH vs. estradiol benzoate plus GnRH to initiate a progesterone-based timed-artificial insemination resynchronization protocol in lactating dairy cows.
Department of Animal Sciences, University of São Paulo, Piracicaba, SP, Brazil, 13418-900Department of Animal and Dairy Sciences, University of Wisconsin-Madison, Madison, WI, USA, 53706
Corresponding author: Roberto Sartori, phone number: +55 19 99777 9258, current mailing address: Department of Animal Science, University of São Paulo, Piracicaba, SP, Brazil, 13418-900
The present study compared 2 strategies to initiate a progesterone (P4)-based timed-artificial insemination (TAI) protocol for lactating dairy cows: only GnRH or estradiol benzoate (EB) plus GnRH. Lactating Holstein cows (n = 487; 184 primiparous and 303 multiparous) from 2 commercial dairy herds were used for their second or greater services postpartum. Weekly nonpregnant cows at pregnancy diagnosis 32 d after a previous AI were randomly assigned to 1 of 2 experimental groups that differed only in the strategy to initiate (d 0) the TAI protocol. On d 0, every cow received a 2.0 g P4 implant, and in Group EB+GnRH, cows were treated with 2.0 mg i.m. of EB and 16.8 µg i.m. of buserelin acetate (GnRH), while in Group GnRH, cows received only 16.8 µg i.m of GnRH. Seven d later (d 7), 0.530 mg i.m. of cloprostenol sodium (PGF) was administered in all cows, followed by a second dose on d 8, concomitant with 1.0 mg i.m. of estradiol cypionate (EC) and P4 implant withdrawal. The TAI was performed on d 10 (48 h after P4 device withdrawal) in both experimental groups. Only conventional Holstein semen was used throughout the study. The percentage of cows with corpus luteum (CL) on d 0 (73%) and overall ovulation rate after d 0 (54%) did not differ between groups. The CL regression between d 0 and the first PGF treatment was greater in EB+GnRH than GnRH group (42 vs. 31%). Consequently, the proportion of cows with CL at PGF was greater when only GnRH was used on d 0 compared with EB plus GnRH (86 vs. 82%), and the mean number of CL at PGF was greater (1.23 vs. 1.11). The expression of estrus near TAI was greater in GnRH group (84 vs. 77%), and cows showing estrus had greater (44 vs. 10%) pregnancy per AI (P/AI) on d 32 for both treatments. There was no effect of the presence of CL on d 0 or at PGF, nor of ovulation after d 0 or CL regression between d 0 and d 7 on fertility. However, fertility was critically impaired when cows did not have CL at both times, d 0 and at PGF treatment. There was no interaction between treatment and other variables, and the P/AI was similar in cows receiving EB plus GnRH or only GnRH on d 0 (37.8 vs. 36.6%). In summary, although there was no detectable difference in P/AI between treatments, this study demonstrated potential negative physiological outcomes caused by EB treatment on d 0 (greater incidence of luteolysis after d 0 and fewer cows with CL at PGF treatment). In conclusion, there was no benefit of adding EB at the initiation of a P4-based TAI protocol on fertility compared with using GnRH alone, despite differences in ovarian dynamics and expression of estrus.
Interpretive Summary Nonpregnant cows must be rapidly re-inseminated in dairy herds, which can be performed by resynchronizing the ovulation when initiating a timed-artificial insemination (TAI) protocol at the nonpregnancy diagnosis. Our study compared two strategies to initiate a progesterone-based Resynch TAI protocol. The results show no benefit of adding estradiol benzoate (EB) at the initiation of the protocol on fertility compared to using GnRH alone, despite differences in ovarian dynamics and expression of estrus.
INTRODUCTION
Timed-artificial insemination (TAI) protocols have been widely used in dairy herds, thereby reducing the need for detection of estrus, improving reproductive efficiency and profitability of dairy operations (
Short communication: Economic impact among 7 reproductive programs for lactating dairy cows, including a sensitivity analysis of the cost of hormonal treatments.
). To increase 21-d pregnancy rate, cows that fail to become pregnant after an AI must be rapidly identified and re-inseminated. To shorten the interbreeding intervals, herds detect estrus for second and greater services, although the success of this strategy relies on the service rate (SR), based on efficiency of the herd in detecting estrus. Alternatively, insemination of nonpregnant cows can be performed after resynchronization of ovulation using TAI protocols (commonly termed Resynch), usually initiated at the time of or before the nonpregnancy diagnosis (NPD;
Effect of timing of initiation of resynchronization and presynchronization with gonadotropin-releasing hormone on fertility of resynchronized inseminations in lactating dairy cows.
Resynchronization of ovulation protocols for dairy cows including or not including gonadotropin-releasing hormone to induce a new follicular wave: Effects on re-insemination pattern, ovarian responses, and pregnancy outcomes.
Effect of timing of initiation of resynchronization and presynchronization with gonadotropin-releasing hormone on fertility of resynchronized inseminations in lactating dairy cows.
Modifications to Ovsynch improve fertility during resynchronization: Evaluation of presynchronization with gonadotropin-releasing hormone 6 d before initiation of Ovsynch and addition of a second prostaglandin F2α treatment.
) or estradiol (E2) plus progesterone (P4)-based protocols, which are initiated with E2 benzoate (EB) and intravaginal P4 implants, sometimes combined with GnRH treatment (
Effect of adding a gonadotropin-releasing-hormone treatment at the beginning and a second prostaglandin F 2α treatment at the end of an estradiol-based protocol for timed artificial insemination in lactating dairy cows during cool or hot seasons of the year.
Follicular dynamics, circulating progesterone, and fertility in Holstein cows synchronized with reused intravaginal progesterone implants that were sanitized by autoclave or chemical disinfection.
Effect of the addition of GnRH and a second prostaglandin F2α treatment on pregnancy per artificial insemination in lactating dairy cows submitted to an estradiol/progesterone-based timed-AI protocol.
). Nevertheless, there are physiological aspects of the E2/P4-based protocols that can impair fertility. For instance, treatment with EB was associated with ∼40% corpus luteum (CL) regression between d 0 and the day of PGF treatment (
Progesterone-based fixed-time artificial insemination protocols for dairy cows: Gonadotropin-releasing hormone versus estradiol benzoate at initiation and estradiol cypionate versus estradiol benzoate at the end.
Increasing estradiol benzoate, pretreatment with gonadotropin-releasing hormone, and impediments for successful estradiol-based fixed-time artificial insemination protocols in dairy cattle.
Follicular dynamics, circulating progesterone, and fertility in Holstein cows synchronized with reused intravaginal progesterone implants that were sanitized by autoclave or chemical disinfection.
Increased fertility in lactating dairy cows resynchronized with Double-Ovsynch compared with Ovsynch initiated 32 d after timed artificial insemination.
Increasing estradiol benzoate, pretreatment with gonadotropin-releasing hormone, and impediments for successful estradiol-based fixed-time artificial insemination protocols in dairy cattle.
). Nevertheless, many veterinarians and producers, especially from South America, continue to use EB combined with GnRH at the initiation due to a lack of studies comparing GnRH alone at the start of the protocol to GnRH plus EB. Thus, the present study compared these 2 strategies, evaluating the potential negative effect of EB treatment on physiological responses and fertility. Two main hypotheses were proposed for the study: 1) cows receiving EB on d 0 of the TAI protocol would have greater incidence of CL regression between d 0 and PGF treatment of the protocol, resulting in fewer cows with CL and a smaller number of CL at the time of PGF, and 2) cows initiating the protocol only with GnRH would have greater fertility than cows that received EB plus GnRH.
MATERIALS AND METHODS
This experiment was conducted in 2 commercial dairy farms, and the Animal Care and Use Committee of the Luiz de Queiroz College of Agriculture of the University of São Paulo (ESALQ/USP) approved all procedures involving cows in this study (protocol # 2017.5.11620.11.3).
Animals, Housing, Herd Management and Diets
In Farm 1, the cows were housed in free-stall barns with ventilation by fans, whereas farm 2 had cross-ventilation facilities. In both farms, cows were milked thrice a day and had free access to water, mineral salt and were fed ad libitum with a TMR based on corn silage as the main forage and concentrate based on corn and soybean meal with minerals and vitamins balanced to meet or exceed the nutritional requirements (National Research Council –
) of lactating dairy cows producing approximately 40 kg/d of milk.
The reproductive management of both farms was 100% TAI for first service and a combination of estrus detection and TAI for re-insemination. Cows not detected in estrus and re-inseminated had their pregnancy diagnosis performed 28 to 34 d post-AI and those not pregnant were enrolled in the TAI protocols used in the experiment. In this the study, cows in farm 1 (n = 260) were enrolled during the hot season of a tropical region (October to March), and farm 2 (n = 227) enrolled cows during an entire year. Lactating Holstein cows (n = 487; 184 primiparous and 303 multiparous) were used for their second (n = 158) or greater services (n = 327) postpartum. At the beginning of the experiment (d 0), cows were at 184.4 ± 102.8 d in milk (DIM ± standard deviation), with body condition score (BCS) of 3.11 ± 0.03 (scale from 1 to 5 according to
Weekly, nonpregnant cows at pregnancy diagnosis 32 d after a previous AI were randomly assigned according to parity and number of AI to 1 of 2 experimental groups (Figure 1) that differed only in the strategy to initiate (d 0) the TAI protocol. On d 0, every cow received a 2.0 g P4 device (Repro sync, GlobalGen, Jaboticabal, Brazil). In Group EB+GnRH, cows were treated with 2.0 mg i.m. of EB (Syncrogen, GlobalGen) and 16.8 µg i.m. of buserelin acetate (GnRH; Maxrelin, GlobalGen), whereas in Group GnRH, cows received only 16.8 µg i.m of GnRH. The higher dose of buserelin was chosen based on previous studies from our laboratory that have shown a decrease in LH peak and ovulation when a regular dose of GnRH is given in the presence of high circulating P4 (
Effect of progesterone from corpus luteum, intravaginal implant, or both on luteinizing hormone release, ovulatory response, and subsequent luteal development after gonadotropin-releasing hormone treatment in cows.
). Seven d later (d 7), 0.530 mg i.m. of cloprostenol sodium (PGF; Induscio, GlobalGen) was administered in all cows, followed by a second dose on d 8, concomitant with 1.0 mg i.m. of estradiol cypionate (EC; Cipion, GlobalGen) and P4 implant withdrawal. The TAI was performed on d 10 (48 h after P4 device withdrawal) in both experimental groups. Within the protocols, all cows were inseminated at the scheduled time without changes due to expression of estrus at the end of the TAI protocol. Only conventional Holstein semen from multiple sires was used during the experimental period and only one technician in each farm performed the AI and this technician was blind to treatment group.
Figure 1Experimental design with hormonal treatments and procedures performed during progesterone (P4)-based timed-artificial insemination (TAI) protocols. On d 0, all nonpregnant cows received a 2.0 g P4 implant. In Group EB+GnRH, cows were treated with 2.0 mg of estradiol benzoate (EB) and 16.8 µg of buserelin acetate (GnRH), while in Group GnRH, cows received only 16.8 µg of GnRH. Seven d later (d 7), 0.530 mg of cloprostenol sodium (PGF) was administered in all cows, followed by a second dose on d 8, concomitant with 1.0 mg of estradiol cypionate (EC) and P4 implant withdrawal. The TAI was performed on d 10 (48 h after P4 device withdrawal) in both experimental groups. Ultrasound evaluations (US) to check presence and number of corpora lutea were performed on d 0 and d 7.
Ovarian Structures, Expression of Estrus, and Pregnancy Diagnosis
To check for presence (independent of its diameter) and number of CL, and to evaluate the response to hormonal treatments, ultrasound evaluations were performed on d 0 and d 7 in all cows. Ovulation was defined as cows that had a follicle greater than 8 mm on d 0 and, subsequently, a new CL was observed on d 7 (time of PGF treatment), similar to
Effect of adding a gonadotropin-releasing-hormone treatment at the beginning and a second prostaglandin F 2α treatment at the end of an estradiol-based protocol for timed artificial insemination in lactating dairy cows during cool or hot seasons of the year.
. Moreover, if the cow had a CL with a fluid-filled cavity on d 7 in the same ovary that had a compact CL on d 0, it was also considered an ovulation. Regression of CL during the protocol (between d 0 and d 7) was determined based on the disappearance of any CL present on d 0. The examination was performed with transrectal ultrasonography of the reproductive tract using an 8–5 MHz multi-frequency linear-array transducer (Ibex Lite, E.I. Medical Imaging, Loveland, CO). For follicle diameter, perpendicular measurements at maximum distances between 2 opposite borders were taken using the built-in calipers of the ultrasound machine. The diameter was determined as the mean of these 2 measures.
Expression of estrus was evaluated using a “rump-mounted patch” (BOViFLAG, Bovitime Animal Products LTD, Stellenbosch, South Africa) placed on d 8 (at the time of P4 withdrawal and EC administration), and cows were considered expressing estrus when the device was activated (e.g., > 50% of the silver scratch-off layer was rubbed off, changing the patch color) by d 10 (time of AI). The evaluation of expression of estrus was performed only on d 10.
Pregnancy per AI (P/AI) diagnosis was performed 32 d (both farms) and 60 d (only on farm 1) after TAI by transrectal ultrasound of the reproductive tract by confirming an embryo heartbeat.
Statistical Analysis
The physiologic measures were sufficiently powered to provide good reliability for detecting differences. However, there was some concern related to the possibility of a Type II error for the binomial data, particularly for the P/AI results. A simple power calculation for a 10% difference (37% vs. 47%) using α = 0.05 and a sample size per group = 238 gave a 0.6 power for a 2-tailed test and of 0.72 for a one-tailed test. There would be sufficient power (≥0.80) to detect differences in P/AI of ≥13% points (i.e., 36% vs. 49%).
Statistical analyses were performed using the Statistical Analysis System (SAS, Version 9.4 for Windows SAS Institute Inc., Cary, NC). Analyses for continuous variables (number of CL at PGF treatment) were performed using the GLIMMIX procedure fitting a Gaussian distribution.
Analyses of binomial variables (presence of CL on d 0 and d 7, ovulation after d 0, CL regression after d 0, expression of estrus, P/AI on d 32 and d 60, and pregnancy loss) were performed using the GLIMMIX procedure fitting a binomial distribution with Logit Link function. Additionally, the option ddfm = kenwardroger was included in the model statement to adjust the degrees of freedom for variances.
The selection of the model that best fit each variable of interest was performed by finding the model with the lowest value for the Akaike Information Criterion Corrected (AICC) using the forward procedure removing variables with P > 0.20 from the model.
For presence of CL on d 0, we studied the effects of treatment, farm, parity, AI number, and BCS on d 0 (≤2.5 or >2.5). The final model included effect of treatment, farm, parity and BCS. Regarding ovulation after d 0, after studying the effects of treatment, farm, parity, BCS, AI number, and presence of CL on d 0, the final model included effects of treatment, BCS and presence of CL. For CL regression during the protocol, the final model included effect of treatment, after evaluating the effects of treatment, parity, farm, AI number, and interaction between treatment and parity. The final model for presence and number of CL on d 7 included effects of treatment, farm, and BCS, although AI number, parity and their interactions with treatment were also studied. A separate model was developed to evaluate the effect of ovulation and CL regression after d 0 on presence of CL on d 7. Regarding expression of estrus, the model included effects of treatment, farm, parity, BCS, and ovulation after d 0. The effects of CL presence on d 0 and d 7, CL regression during the protocol, and interaction between treatment and parity were also evaluated.
To better understand the effect of experimental treatments on CL dynamics during the protocol, 4 categories of cows were created (Figure 2): absence of CL on d 0 and d 7; absence of CL on d 0 and presence on d 7; presence of CL on d 0 and absence on d 7; and presence of CL on d 0 and d 7. Regarding classes of cows according to presence of CL on d 0 and d 7, the final model included effects of treatment, farm, and parity, although BCS and interaction between treatment and parity were also studied.
Figure 2Proportion of cows with or without corpus luteum (CL) on d 0 and at the time of PGF treatment based on the strategy to initiate the timed-artificial insemination protocol. Treatments were estradiol benzoate plus GnRH (EB+GnRH) or only GnRH (GnRH) on d 0. The dose of GnRH was 16.8 µg of buserelin acetate and the dose of EB was 2 mg.
The final model for P/AI on d 32 included effects of treatment, BCS, parity, farm, AI number, CL on d 0, ovulation after d 0, and the interaction between CL and ovulation after d 0. Effects of presence of CL on d7 and interaction between treatment with parity, CL on d0 and AI number were also studied. Separate models were built to evaluate the effects of expression of estrus, classes of cows according to CL on d 0 and d 7, and the interaction between ovulation and CL regression after d 0. The model for P/AI on d 60 only included data from Farm 1 and included effects of treatment and expression of estrus and the model for pregnancy loss included only treatment effect.
Tukey honest significant difference post hoc test was performed to determine differences. Values are presented as means ± standard error of mean (SEM) for continuous variables and as percentage (%) for binomial variables. Significant differences were declared when P ≤ 0.05, whereas tendencies were considered for 0.05 < P ≤ 0.10.
RESULTS AND DISCUSSION
This research was conducted to evaluate both the physiological and fertility responses to only GnRH compared with GnRH plus EB at the start of a synchronization of ovulation protocol. Since all cows were evaluated for some specific physiological endpoints, these responses had greater statistical power than many previous studies, however, the fertility responses may have been less powered than desired and is subject to a Type II error if there is less than a 12% difference in P/AI.
Effect of Treatment on Ovarian Dynamics, Expression of Estrus and Fertility
On d 0, the overall proportion of nonpregnant cows with CL detected by US was 73.3% (357/487, Table 1), similar to studies that reported about 30% of cows without CL at the time of pregnancy diagnosis (
Effect of an Ovsynch56 protocol initiated at different intervals after insemination with or without a presynchronizing injection of gonadotropin-releasing hormone on fertility in lactating dairy cows.
Adding a second prostaglandin F2alpha treatment to but not reducing the duration of a PRID- Synch protocol increases fertility after resynchronization of ovulation in lactating Holstein cows.
). There was no effect of EB treatment (P = 0.91) on the ovulatory response after d 0, and the overall outcome was 53.6% (261/487), as shown in Table 1, which was greater than the 30–40% achieved in other studies (
Increased fertility in lactating dairy cows resynchronized with Double-Ovsynch compared with Ovsynch initiated 32 d after timed artificial insemination.
Progesterone-based fixed-time artificial insemination protocols for dairy cows: Gonadotropin-releasing hormone versus estradiol benzoate at initiation and estradiol cypionate versus estradiol benzoate at the end.
Comparison of 2 protocols to increase circulating progesterone concentration before timed artificial insemination in lactating dairy cows with or without elevated body temperature.
). This result may be explained by the higher dosage of GnRH or the type of GnRH analog (i.e., buserelin acetate) used in our study. Other studies comparing doses of buserelin and gonadorelin reported that doubling the dosage of GnRH approximately doubled the peak of the LH surge (
Effect of progesterone on magnitude of the luteinizing hormone surge induced by two different doses of gonadotropin-releasing hormone in lactating dairy cows.
Treatments were estradiol benzoate plus GnRH (EB+GnRH) or only GnRH (GnRH) on d 0 of progesterone-based TAI protocols. The dose of GnRH was 16.8 μg of buserelin acetate and the dose of EB was 2 mg.
P-value
EB+GnRH
GnRH
Cows with CL on d 0, % (n/n)
73.5 (175/238)
73.1 (182/249)
0.98
Ovulation after d 0, % (n/n)
53.8 (128/238)
53.4 (133/249)
0.91
CL regression after d 0, % (n/n)
42.3 (74/175)
31.3 (57/182)
0.03
Cows with CL at PGF, % (n/n)
81.9 (195/238)
86.4 (215/249)
0.05
Number of CL at PGF, n (n)
1.11 (238)
1.23 (249)
0.07
Cows with 2 or more CL (%)
16.8% (40/238)
22.1 (55/249)
0.14
Expression of estrus, % (n/n)
76.9 (183/238)
83.5 (208/249)
0.04
1 Treatments were estradiol benzoate plus GnRH (EB+GnRH) or only GnRH (GnRH) on d 0 of progesterone-based TAI protocols. The dose of GnRH was 16.8 μg of buserelin acetate and the dose of EB was 2 mg.
Our hypothesis that more cows receiving EB would have luteolysis between d 0 and d 7 of the protocol was confirmed, as the Group EB+GnRH had fewer cows with CL at the time of PGF and tended to have fewer CL at time of PGF (Table 1). Our data regarding luteolysis were similar to the observation of ∼40% CL disappearance after EB treatment reported in previous studies (
Increasing estradiol benzoate, pretreatment with gonadotropin-releasing hormone, and impediments for successful estradiol-based fixed-time artificial insemination protocols in dairy cattle.
Progesterone-based fixed-time artificial insemination protocols for dairy cows: Gonadotropin-releasing hormone versus estradiol benzoate at initiation and estradiol cypionate versus estradiol benzoate at the end.
). Nevertheless, both groups continued to have more than 80% of the cows with CL at PGF (Table 1), mostly due to the high ovulation (>50%) after GnRH treatment on d 0.
The treatments affected the proportion of cows within the 4 categories created according to the presence of CL on d 0 and at PGF treatment (Figure 2). The treatment only with GnRH on d 0 decreased the proportion of cows with CL on d 0 but no CL at PGF treatment and increased the percentage of cows with CL at both time points (Figure 2). These results can be explained by the greater incidence of luteolysis between d 0 and d 7 in cows from group EB+GnRH compared with only GnRH, with EB treatment decreasing the number of cows with CL at PGF. A previous study comparing EB vs. GnRH on d 0 of TAI protocols performed the same type of analysis and reported greater percentage of cows with CL at the time of PGF treatment when GnRH was used compared with EB (77.3 vs. 58.3%;
Progesterone-based fixed-time artificial insemination protocols for dairy cows: Gonadotropin-releasing hormone versus estradiol benzoate at initiation and estradiol cypionate versus estradiol benzoate at the end.
The proportion (∼80%) of cows expressing estrus near TAI in our study was similar to what was reported for TAI protocols using EC as an ovulation inducer (
Effect of adding a gonadotropin-releasing-hormone treatment at the beginning and a second prostaglandin F 2α treatment at the end of an estradiol-based protocol for timed artificial insemination in lactating dairy cows during cool or hot seasons of the year.
Expression of estrus improves fertility and decreases pregnancy losses in lactating dairy cows that receive artificial insemination or embryo transfer.
). Interestingly, the cows receiving only GnRH on d 0 had greater expression of estrus (Table 1). In the GnRH group, cows ovulating to the d 0 GnRH are expected to have follicular wave emergence earlier in the protocol, between 1 and 2 d after treatment (
Follicular dynamics, circulating progesterone, and fertility in Holstein cows synchronized with reused intravaginal progesterone implants that were sanitized by autoclave or chemical disinfection.
). This should result in follicles with ∼6 d of dominance at AI, thus providing adequate follicular size and E2 production to stimulate expression of estrus in most cows. Cows that did not ovulate after d 0 in the GnRH group could be at the beginning of a follicular wave, in which they would also have follicles with 6 or more days of dominance at the end of the protocol. Conversely, in the EB+GnRH group, cows that ovulate to the GnRH treatment would be expected to have similar follicular dynamics as the GnRH only group. However, the cows in the EB+GnRH group that did not ovulate to the GnRH are likely to have atresia of the follicles with later follicular wave emergence, between d 3 and 5 of the protocol (
Increasing estradiol benzoate, pretreatment with gonadotropin-releasing hormone, and impediments for successful estradiol-based fixed-time artificial insemination protocols in dairy cattle.
). This can be observed in the small but significant difference in expression of estrus (76.9 vs 83.5%) between the treatment groups.
Ovulation to the GnRH treatment at d 0 (Table 2) was not altered by farm or parity with both farms and both primiparous and multiparous cows having ∼53% ovulation to the GnRH. There was an effect of BCS with cows with BCS ≤2.5 having 10.3 percentage points lower ovulation than cows with BCS >2.5. No interaction between BCS and treatment was observed (P > 0.05). The presence of a CL had the greatest effect on ovulation to the d 0 GnRH with ∼30 percentage points reduction in cows that ovulated (Table 2). No interaction between presence of CL on d 0 and treatment was observed (P > 0.05). It was expected that cows with a CL would have a lower ovulation since it is established that the presence of CL and/or high circulating P4 concentrations at the time of GnRH treatment decreases LH peak and ovulatory response (
Effect of progesterone on magnitude of the luteinizing hormone surge induced by two different doses of gonadotropin-releasing hormone in lactating dairy cows.
Feedback effects of estradiol and progesterone on ovulation and fertility of dairy cows after gonadotropin-releasing hormone-induced release of luteinizing hormone.
Effect of progesterone from corpus luteum, intravaginal implant, or both on luteinizing hormone release, ovulatory response, and subsequent luteal development after gonadotropin-releasing hormone treatment in cows.
Table 2Ovulation after d 0 of timed-artificial insemination protocols initiated with GnRH or EB+GnRH divided by farm, parity, BCS and presence of corpus luteum (CL)
The rationale for the experimental design used in this study was based on previous results from the scientific literature and studies performed in our laboratory. When TAI protocols that were initiated with EB or EB plus GnRH were compared, it was reported that the inclusion of GnRH improved fertility of lactating dairy cows, especially in those without CL (
Effect of adding a gonadotropin-releasing-hormone treatment at the beginning and a second prostaglandin F 2α treatment at the end of an estradiol-based protocol for timed artificial insemination in lactating dairy cows during cool or hot seasons of the year.
). In addition, a study from our laboratory compared EB vs. GnRH on d 0 and found that GnRH treatment produced a better P4 milieu during follicle development and tended to have greater fertility (
Progesterone-based fixed-time artificial insemination protocols for dairy cows: Gonadotropin-releasing hormone versus estradiol benzoate at initiation and estradiol cypionate versus estradiol benzoate at the end.
). Interestingly, both studies used gonadorelin and reported low ovulatory responses after GnRH treatment (36%), but still observed better physiological and fertility results when GnRH was used at the beginning of the TAI protocols. A recent compilation of studies that compared protocols initiated only with EB to protocols that used GnRH at the start of the protocol (with or without EB) found that cows that received EB alone had a lower percentage of cows with CL at PGF and lower fertility than cows receiving GnRH (
). Nevertheless, to our knowledge, no previous study has evaluated the effects of combining the EB treatment with GnRH compared with GnRH alone at the beginning of the TAI protocol. One limitation of our study was the use of a 7-d interval between ultrasound examinations to determine ovulation and CL regression, potentially confounding interpretation of our results.
The P/AI on d 32 after TAI was similar between treatments (Table 3), with no interaction between treatment and parity (Table 3). Considering only farm 1, EB+GnRH and GnRH groups produced similar P/AI on d 32 after TAI (35.1 [46/131] vs. 31.8% [41/129]; P = 0.56) and d 60 after TAI (29.0 [38/131] vs. 28.7% [37/129]; P = 0.91) and there was no effect of treatment on pregnancy loss between d 32 and d 60 after TAI (17.4 [8/46] vs. 9.8% [4/41]; P = 0.31).
Table 3Pregnancy per artificial insemination (P/AI) on d 32 after AI based on the strategy to initiate the timed-AI (TAI) protocol
Treatments were estradiol benzoate plus GnRH (EB+GnRH) or only GnRH (GnRH) on d 0 of TAI protocols. The dose of GnRH was 16.8 μg of buserelin acetate, and the dose of EB was 2 mg.
T = effect of treatment; P = effect of parity; and TxP = interaction between treatment and parity.
EB+GnRH
GnRH
T
P
TxP
All cows
37.8 (90/238)
36.6 (91/249)
0.86
—
—
Parity
Primiparous
40.2 (37/92)
37.0 (34/92)
0.86
0.88
0.56
Multiparous
36.3 (53/146)
36.3 (57/157)
1 Treatments were estradiol benzoate plus GnRH (EB+GnRH) or only GnRH (GnRH) on d 0 of TAI protocols. The dose of GnRH was 16.8 μg of buserelin acetate, and the dose of EB was 2 mg.
2 T = effect of treatment; P = effect of parity; and TxP = interaction between treatment and parity.
Thus, our hypothesis that cows receiving EB on d 0 would have lower fertility was not confirmed. As mentioned, EB+GnRH group had greater incidence of luteolysis during the protocol and fewer cows with CL at the time of PGF than cows receiving only GnRH (Table 1). Nevertheless, the fertility was not lower. The lack of a negative effect of EB on d 0 on fertility could be partially explained by the relatively high ovulation obtained in this study (53%), which resulted in more than 80% of cows with CL at the time of PGF, even in EB+GnRH group (Table 1). Other researchers have reported that inclusion of GnRH on d 0 of a TAI protocol initiated with EB increased circulating P4 concentrations and percentage of cows with CL at PGF compared with cows receiving only EB on d 0 (
Effect of adding a gonadotropin-releasing-hormone treatment at the beginning and a second prostaglandin F 2α treatment at the end of an estradiol-based protocol for timed artificial insemination in lactating dairy cows during cool or hot seasons of the year.
). In addition, cows that ovulated at the beginning of TAI protocols will have a CL present at PGF, increased circulating P4 during the protocol, and this could possibly improve fertility, particularly in cows with low circulating P4 or without CL at the onset of the protocol (
Increased fertility in lactating dairy cows resynchronized with Double-Ovsynch compared with Ovsynch initiated 32 d after timed artificial insemination.
Follicular dynamics, circulating progesterone, and fertility in Holstein cows synchronized with reused intravaginal progesterone implants that were sanitized by autoclave or chemical disinfection.
Another aspect that could reduce the negative effect of the CL regression induced by EB treatment is that all cows received a 2.0 g P4 device during the protocol. It is reported that insertion of 2 P4 devices (1.38 g each) did not produce similar circulating P4 concentrations as a mature CL (
Effect of interval between induction of ovulation and artificial insemination (AI) and supplemental progesterone for resynchronization on fertility of dairy cows subjected to a 5-d timed AI program.
). Therefore, in the EB+GnRH group, those cows undergoing luteolysis during the protocol may have benefited from the use of a 2.0 g P4 device in the present study.
Effect of Ovarian Dynamics and Expression of Estrus on Fertility
The effect of several variables on P/AI are shown in Table 4. There were no interactions between treatments and these variables on P/AI (not shown). There was no effect of presence of CL on d 0 or on d 7 on P/AI and there was no effect of number of CL on d 7 on P/AI (Table 4). It has been reported that presence of CL and circulating P4 concentration at the beginning of TAI protocols or at PGF are positively associated with fertility in lactating dairy cows submitted to both Ovsynch-type and E2 plus P4-based protocols (
Increased fertility in lactating dairy cows resynchronized with Double-Ovsynch compared with Ovsynch initiated 32 d after timed artificial insemination.
Progesterone-based fixed-time artificial insemination protocols for dairy cows: Gonadotropin-releasing hormone versus estradiol benzoate at initiation and estradiol cypionate versus estradiol benzoate at the end.
Comparison of 2 protocols to increase circulating progesterone concentration before timed artificial insemination in lactating dairy cows with or without elevated body temperature.
Luteal Presence and Ovarian Response at the Beginning of a Timed Artificial Insemination Protocol for Lactating Dairy Cows Affect Fertility: A Meta-Analysis.
). In a previous study, cows with CL at the time of PGF had a P/AI of 40% (418/1,045) on d 30 compared with 24% (133/551) attained by cows without CL (
Comparison of 2 protocols to increase circulating progesterone concentration before timed artificial insemination in lactating dairy cows with or without elevated body temperature.
). Similarly, presence of an active CL or circulating P4 ≥ 1.0 ng/mL at the time of PGF resulted in greater fertility of resynchronized cows (37 vs. 8%;
Increased fertility in lactating dairy cows resynchronized with Double-Ovsynch compared with Ovsynch initiated 32 d after timed artificial insemination.
). Two aspects of the present study may have contributed to the lack of effect of CL at PGF on fertility. First, almost all cows (84.2%; 410/487) had a CL at the time of PGF, and second all cows in the study received a 2.0 g P4 device during the protocol and both of these factors would have increased circulating P4 concentrations during development of the preovulatory follicle and made it less likely to detect differences in P/AI.
Table 4Pregnancy per artificial insemination (P/AI) 32 d after timed-AI according to ovarian dynamics and expression of estrus
To better understand the impact of the presence of CL during the protocol on fertility, the P/AI was evaluated based on the 4 CL categories, created by the presence or absence of CL on d 0 and at PGF (Figure 3). Cows without CL at the beginning and at the time of PGF had the lowest P/AI on d 32 after TAI and it was 43% lower than cows in the other 3 groups which were not different from each other (38.2%; 174/455). This result reinforces the importance of high circulating P4 concentrations during follicular development on fertility of lactating dairy cows. Two other studies that performed similar analyses also reported lower fertility in cows without CL compared with cows with CL during the protocol, especially in those without CL at both the beginning and at PGF treatment (
Progesterone-based fixed-time artificial insemination protocols for dairy cows: Gonadotropin-releasing hormone versus estradiol benzoate at initiation and estradiol cypionate versus estradiol benzoate at the end.
Comparison of 2 protocols to increase circulating progesterone concentration before timed artificial insemination in lactating dairy cows with or without elevated body temperature.
Figure 3Pregnancy per artificial insemination (P/AI) 32 d after timed-AI (TAI) in cows with or without corpus luteum (CL) on d 0 and at the time of PGF treatment of TAI protocols.
There was no interaction (P > 0.10) between ovulation after d 0 and treatment or parity on fertility. In addition, ovulating after d 0 did not influence P/AI (Table 4). These results contrast with data from a study that reported greater fertility in cows ovulating at the beginning of the TAI protocol (
Comparison of 2 protocols to increase circulating progesterone concentration before timed artificial insemination in lactating dairy cows with or without elevated body temperature.
). To further explore this effect, results for P/AI were divided by cows that had or did not have a CL at the beginning of the protocol and then by whether they ovulated to GnRH (Table 5). Ovulation increased P/AI by 77% in cows without CL at the beginning of the protocol but had no effect on P/AI in cows with a CL (Table 5). The positive effect of ovulation in cows without CL has been previously reported (
Increased fertility in lactating dairy cows resynchronized with Double-Ovsynch compared with Ovsynch initiated 32 d after timed artificial insemination.
Follicular dynamics, circulating progesterone, and fertility in Holstein cows synchronized with reused intravaginal progesterone implants that were sanitized by autoclave or chemical disinfection.
Another intriguing result obtained in the present study was that luteolysis during the protocol (between d 0 and PGF treatment) did not impact fertility (37.4 [49/131] vs. 38.5% [87/226] for cows with and without luteolysis after d 0, respectively). This lack of effect could be due to an elevation in circulating P4 in most cows during the protocol due to high ovulation incidence after d 0, high proportion of cows with CL at the time of PGF, and the presence of the 2.0 g P4 implant in all cows. We expected that ovulation after d 0 might have improved fertility in cows undergoing lysis of the pre-existing CL during the protocol. However, there was no impact of ovulation after d 0 on P/AI of cows undergoing luteolysis (38.0 [27/71] vs. 36.7% [22/60]; P = 0.82). The lack of a positive effect of ovulation on d 0 in cows undergoing luteolysis during the protocol may be related to the low percentage of cows without a CL on d 7 (26%) and the presence of the P4 device (
In our study, expression of estrus did not interact with experimental treatments, but dramatically impacted fertility, with cows expressing estrus having 4.2 times greater P/AI (Table 4). It is reported that expression of estrus at the end of TAI protocols increases fertility (
Expression of estrus improves fertility and decreases pregnancy losses in lactating dairy cows that receive artificial insemination or embryo transfer.
) with a reported increase of 13 percentage points in P/AI at d 30 and d 60 after AI in 5,430 cows submitted to TAI protocols with EC as the ovulation inducer. In addition, it is likely that there was a lack of ovulation to the protocol in many of the cows that did not show estrus, particularly since they were given EC as the ovulation inducer.
Effect of Farm, BCS, and Number of AI on Fertility
There was a farm effect on fertility, in which farm 2 achieved 8 percentage points greater P/AI (41.4 [94/227] vs. 33.5% [87/266]; P = 0.03). This difference might be expected since farm 2 had better overall management and better comfort for the cows. The use of only 2 farms with different management methods may be considered a limitation of the study; although there was no treatment by farm interaction. In addition, it is well established that thinner cows have decreased fertility compared with cows with BCS >2.5 (
). Our results support the effect of BCS, with cows with lower BCS (≤2.5) having 37% lower fertility than cows with BCS >2.5 (25.3 [24/95] vs. 40.1% [157/392]; P = 0.008). Similar to other studies (
Effect of timing of initiation of resynchronization and presynchronization with gonadotropin-releasing hormone on fertility of resynchronized inseminations in lactating dairy cows.
), cows receiving the second and third AI had greater fertility than those receiving ≥4 AI (41.1 [65/158]a, 41.4 [36/87]a and 33.1% [80/242]b, respectively; P = 0.05).
CONCLUSIONS
In conclusion, the results of this study demonstrate that TAI protocols that are initiated with EB plus GnRH or only GnRH produced similar fertility. Even though treatment with EB produced greater incidence of luteolysis between d 0 and d 7 of the protocol, resulting in fewer cows with CL at the time of PGF treatment, there was no detectable difference between treatments on P/AI. Thus, there seems to be no fertility benefit or detriment of adding EB at the initiation of a resynchronization, P4-based TAI protocol in lactating dairy cows compared with using GnRH alone, despite differences in ovarian dynamics and expression of estrus.
ACKNOWLEDGMENTS
The authors thank the owners and staff of farms São Jorge and Céu Azul for the use of their cows and facilities. We appreciate the Coordination for the Improvement of Higher Education (CAPES, Brasília, Brazil) and Brazilian National Council for Scientific and Technological Development (CNPq, Brasília, Brazil) for the financial support for the experiment and students that worked during the experiment. We also thank the São Paulo Research Foundation (FAPESP, São Paulo, Brazil) for the support in the study by the scholarship grant # 2017/15904-3 and fund grant # 2018/03798-7. Our appreciation is extended to GlobalGen vet science (Jaboticabal, São Paulo, Brazil) for providing all hormones used in the TAI protocols.
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