A single dose of FSH or hCG during a split-time AI program did not enhance follicular growth or pregnancy per artificial insemination in beef heifers

ABSTRACT The objective of this study was to evaluate the effect of a low dose of hCG or FSH on follicle growth and pregnancy per artificial insemination (AI) in crossbreed beef heifers subjected to a split-time AI program. All heifers (n = 386) were subjected to a 7-d Co-synch protocol and an intravaginal controlled internal drug release (CIDR) device (GnRH and CIDR insertion; 7 days later PGF2α, CIDR removal, and application of a tail head estrus detection patch; 36 hours later AI of heifers detected in estrus, 24 hours later timed AI of remaining heifers). At CIDR removal, heifers were randomly assigned to receive either 150 IU of hCG, 20 mg of FSH, or remained as untreated controls. The diameter of the preovulatory follicle and the pregnancy per AI did not differ among treatments. A smaller (P = 0.035) proportion of heifers in the hCG group were detected in estrus compared with control. Additionally, the incidence of premature ovulation was greater (P < 0.01) among heifers treated with hCG compared with control. In conclusion, using a low dosage of hCG or FSH at the time of device removal did not affect pre-ovulatory follicle growth or the proportion of pregnant heifers.


Introduction
The primary purposes of using estrus synchronization protocols in cow-calf operations are to concentrate calvings at the beginning of the calving season and increase calf uniformity, leading to greater weaning weights and improving profitability (Baruselli et al. 2012(Baruselli et al. , 2018;;Bó and Baruselli 2014).When estrus synchronization is combined with artificial insemination (AI) or timed artificial insemination (TAI), the introduction of new and better genetics to the herd is also a major advantage.However, despite such benefits, the last report of the USDA regarding cow-calf operation management practices in the USA showed that only 11.6% of producers use AI and 7.3% use estrus synchronization (USDA 2020).
The Co-synch estrus synchronization protocol and its variations are GnRH-based and used worldwide (Beef reproduction task force 2021; Lamb and Mercadante 2016).Pregnancy per artificial insemination (P/AI) remains variable when using the Co-Synch protocols, ranging from 42 to 67% and 39% to 68% for cows and heifers, respectively (Martínez et al. 2002;Bridges et al. 2008;Kasimanickam et al. 2008Kasimanickam et al. , 2020;;Marquezini et al. 2011;Whittier et al. 2013;Madureira et al. 2020).The use of exogenous gonadotropins at the time of intravaginal progesterone (P 4 ) device removal to enhance follicular growth by supporting the low release of luteinizing hormone (LH) has become a common adaptation in estradiol-based protocols in South America, for Bos taurus indicus cattle (Baruselli et al. 2012) and for Bos taurus cattle (Mínguez and Calvo 2020).Equine chorionic gonadotropin (eCG) (Bó and Baruselli 2014;Prata et al. 2018), human chorionic gonadotropin (hCG) (Santos et al. 2001;Prata et al. 2018;Souza et al. 2019), and follicle-stimulating hormone (FSH) (Santos et al. 2007) have shown beneficial results in P/AI when used at different moments in estradiol-based protocols.However, the use of hCG at the time of intravaginal P 4 device removal has not been explored in GnRH-based protocols and only Small et al. 2008 have reported the use of FSH at the time of intravaginal P 4 device removal in GnRH based protocols.
The eCG, which has an FSHand LH-like actions in cattle, has been shown to increase P/AI from 61.5 to 71.7% (Sales et al. 2011).However, eCG is not commercially available for estrus synchronization protocols in the USA (U.S. Food and Drug Administration 2019).Therefore, exogenous FSH is an alternative strategy that has been shown to increase P/AI from 29.9 to 58.5% when applied during estradiol-based protocols (Santos et al. 2007).Exogenous FSH has been widely used for superovulation protocols in beef and dairy cattle due to its effect on FSH receptors in the granulosa cells, enhancing development of multiple preovulatory follicles allowing more follicles to continue growing past deviation (Bó and Mapletoft 2020).In addition, it has also been used in AI protocols to improve P/AI results.Santos et al. 2007 evaluated the use of 10 or 20 mg of FSH or 400 IU of eCG at the time of the intravaginal P 4 device withdrawal during an estradiol-based protocol in Bos taurus indicus cows.The authors showed an increase in P/AI using either gonadotropin compared to the control group (eCG = 60.2%; 10 mg of FSH = 59.7%; 20 mg of FSH = 58.5%;Control = 29.9%).Both doses of FSH and eCG presented similar P/AI results.In contrast, Sales et al. 2011 reported that supplementing cows with 10 mg of FSH at the time of intravaginal P 4 device removal did not increase P/AI compared with controls and eCG (10 mg of FSH = 60.0%;eCG = 71.6%;Control: 61.5%).
Alternatively, hCG is another gonadotropin hormone that can enhance follicular growth and P/AI by inducing ovulation, because of its LH-like effect.The main use of hCG has been to treat cystic cows as it has a similar therapeutic effect as GnRH (De Rensis et al. 2010).Besides, hCG has been used after AI to promote ovulation and formation of an accessory corpus luteum (CL).Santos et al. 2001 showed that after applying 3,300 IU of hCG in dairy cattle five days after AI, plasma progesterone levels increased (Control: 14.8 ng/mL; hCG: 20.7 ng/ ml) associated with an increased number of CLs.The hCG has been tested in AI protocols as an ovulation inductor, but results were not satisfactory when used in GnRH-based protocols.Research conducted in beef cattle mainly attempted to replace GnRH with hCG in a Co-Synch protocol.Burns et al. 2008 showed that using 1,000 IU of hCG to replace GnRH on day 0 of the estrus synchronization protocol and TAI day decreased the P/AI (GnRH: 53.4% vs. hCG: 38.9%).Geary et al. 2001 replaced GnRH with 2,500 IU of hCG on day 0 and at TAI in cows with or without calf removal for 48 h, and no difference was found between the four treatments (GnRH without calf removal: 49%; GnRH with calf removal: 46%; hCG without calf removal: 34%; hCG with calf removal: 35%; P = 0.44).Recently, a new strategy of using a low dose of hCG (150 IU) at the time of the intravaginal P 4 device removal was described by Souza et al. 2019 using estradiol-based protocols.They found an increase in the P/AI (Control = 43.3% vs. hCG = 53.2%).That study suggested that hCG may provide LH-like stimulus to final stages of follicular growth to ultimately enhance P/AI.However, the effect of hCG to stimulate final follicular growth in GnRH-based protocols has not been tested.
We hypothesize that a small dose of FSH (20 mg) or hCG (150 IU) at the time of the intravaginal P 4 device removal enhances follicular growth and P/AI in crossbred beef heifers subjected to a 7-d Co-synch + CIDR protocol.

. Material and methods
The experiment was conducted with the approval of the Institutional Animal Care and Use Committee of the University of Florida (Study number 202011004).

Animals
A total of 386 commercial crossbred yearling (approx.12-16 month of age) heifers (Bos taurus indicus x Bos taurus taurus) from a commercial farm in Okeechobee, Florida, were enrolled in this experiment during a 90-day breeding season.Heifers were kept on Bahiagrass (Paspalum notatum) and Limpograss (Hemarthria altissima) pastures, supplemented with a mixed ration feed, a mineral salt, and ad libitum access to water.For management purposes, heifers were divided into two groups (G1 n = 197; G2 n = 189).
Heifers were randomly assigned to three treatments within groups (Control n = 134, FSH n = 124, and hCG n = 128) and were allocated to treatments homogeneously, according to reproductive tract score (RTS; details in ultrasonography section; [Anderson et al. 1991]), body condition score (BCS; 1 to 9 scale; [Wagner et al. 1988]) and pubertal status (cycling: the presence of a corpus luteum, or not cycling: absence of a corpus luteum).Data was organized in an Excel spreadsheet, and heifers were ranked according to the three criteria.Next, a random number was generated for each heifer in Excel, which was used to allocate them to one of the treatments.We wanted to be sure that each treatment received homogenous groups of heifers and that each heifer had the same chance to receive any treatment.Three different sires were used for artificial insemination, and sires were randomly assigned to heifers within the treatments.

Reproductive management
All heifers were subjected to a 90-d breeding season.Estrus in all heifers were synchronized with a 7-d Co-synch + CIDR (controlled internal drug release; Zoetis) protocol (Figure 1).On day 0, heifers were treated with an intramuscular (IM) injection of 100 µg of GnRH (Factrel, Zoetis) and an intravaginal CIDR device containing 1.38 g of progesterone (Eazi-Breed CIDR, Zoetis) was inserted.On day 7, the intravaginal CIDR device was removed in the afternoon, an injection of 25 mg of dinoprost tromethamine (Prostaglandin (PGF); Lutalyse HighCon Injection, Zoetis) was given IM, and an estrus detection patch (Estrotect™) was placed halfway between the hip and tail head on all heifers.Additionally, heifers in the FSH group received an IM injection of 20 mg of FSH (Folltropin, Vetoquinol).Heifers in the hCG group were treated with an IM injection of 150 IU of hCG (Chorulon, Merck).Estrus detection was carried out 36 hours after the intravaginal P 4 device removal.Heifers that presented more than 50% of the estrus detection patch rubbed off were considered in estrus and were artificially inseminated 12 hours later and received an IM injection of 100 µg of GnRH.On day 10, the heifers that presented estrus after 36 hours and heifers that did not present estrus were TAI, 60 hours after intravaginal P 4 device removal.All heifers received an IM injection of 100 µg of GnRH at the time of the TAI.Ten days after TAI, the heifers were exposed to bulls for natural service in a ratio of 1 bull per 20 heifers.

Ultrasonography
Measurements of pre ovulatory follicles (POF) were performed on a single, still sonogram of the apparent maximal area for each structure.(Esaote ultrasound, MyLab Delta Vet, with 10-5 MHz transducer).On day 0, presence of CL and RTS on a scale from 1 to 5 (1: no follicles, no CL immature uterine horn; 2: the presence of 8 mm follicles, uterine horn of 20-25 mm diameter, no tone; 3: presence of 8-10 mm follicles, uterine horn of 25-30 mm diameter and slight tone; 4: presence of > 10 mm follicles, 30 mm diameter and good tone; 5: presence of > 10 mm follicles and corpus luteum, > 30 mm diameter and good tone; [Anderson et al. 1991]) were determined.On day 7 and at AI (either 12 hours after estrus detection or TAIday 10), the length (L) and width (W) of the dominant follicle were measured to calculate the follicular diameter (D).The follicular diameter was calculated with the formula: The follicular growth was calculated by subtracting the follicle diameter at AI from the follicle diameter on day 7. Premature ovulation was defined for heifers with a deviated follicle (>5 mm; Cooke et al. 2020) on day 7 but without a dominant preovulatory follicle at AI. P/AI and P/AI + natural service (P/ AI + NS) were evaluated 30 and 130 days after TAI, respectively.Heifers were considered pregnant if a viable (i.e.presence of heartbeat) embryo or fetus was detected.

Statistical analyses
Dependent binary variables (P/AI, NS, P/A + NS, estrus detection, and premature ovulation) were analyzed using the GLIMMIX procedure of SAS (SAS Institute, Cary, NC).When a significant effect was found, multiple comparison tests were performed.
The complete model for P/AI accounted for the following fixed independent variables: treatment (Control, FSH, or hCG), follicular growth rate, cyclicity status, BCS, estrus detection and sire.Variables were removed from the model by backward elimination if P > 0.10.Treatment was forced in the final model.Therefore, the final model for P/AI (n = 386), NS (n = 241) and P/ AI + NS (n = 386) considered the treatment effect only.The model to evaluate estrus response accounted for treatment effect only and a total of 19 heifers were not included in the analysis because they lost their Estrotect patch.The complete model for premature ovulation accounted for treatment and follicle diameter on day 7.
Dependent continuous variables (follicle diameter on day 7 and at AI, and follicular growth rate) were analyzed by ANOVA using the GLM procedure of SAS.Follicle diameter at day 7 was used as a covariate when follicle diameter at AI and follicular growth rate was analyzed.In addition, continuous data were tested for normality of the residues (Shapiro -Wilk test) and homogeneity of variances (Levene's and Bartlett's tests) and transformed when necessary.The final model for follicle diameter on day 7 accounted for the fixed independent variable of treatment only.For the follicle diameter at AI and follicular growth rate the final model accounted for the fixed independent variable of treatment and follicle diameter on day 7.One heifer was not included in the analyses of follicle diameter at day 7 and AI day because she was not scanned.Forty- one heifers were not included in the follicle diameter at AI day as they ovulated before, also six heifers were removed as they did not have a measurable follicle.
We conducted a post hoc power analysis for follicle diameter on day seven and pregnancy per AI (using the PROC POWER procedure of SAS software).Both follicle diameter at day seven and pregnancy per AI had a high power (> 99% and 92%, respectively).P-values are considered significant at <0.05 and tendencies at <0.10.

Results
The heifers in the current experiment had on average a BCS of 5, a RTS of 4 and 90% were cycling at the time the P 4 intravaginal device was inserted.

. Estrus detection
A total of 148 (Control n = 57, FSH n = 57, and hCG n = 34) heifers presented estrus 36 hours after removing the intravaginal CIDR.The proportion of heifers detected in estrus decreased (P = 0.03) for hCG compared with control and FSH groups (Table 1).
The proportion of heifers with premature ovulation was greater (P < 0.01) in the hCG group when compared with the control group, while the FSH group was similar to both the other groups (Control = 3.42%; FSH = 12.18%; hCG = 17.53%;P ≤ 0.01; Table 1).
Independent of the treatments, heifers that were inseminated 12 hours after estrus detection had larger follicle diameters on day 7 and at AI day when compared to heifers that were submitted to TAI at day 10 (Table 2).Cyclicity status at the beginning of the protocol did not influence follicle diameters on day 7 and at AI day (Table 3).
Independently of the treatments, P/AI and NS + P/AI were not influenced by the AI day (12 hours after estrus detection or TAI at day 10; Table 2) or the cyclicity status at the beginning of the protocol (Table 3).

. Discussion
Estrus synchronization protocols in beef heifers continue to yield highly variable P/AI, ranging from 39 to 65% (Martínez et al. 2002;Kasimanickam et al. 2020;Madureira et al. 2020).Bos taurus indicus influenced cattle have benefited from the administration of exogenous gonadotropins at the time of the intravaginal P 4 device removal to enhance follicular  growth and P/AI (Baruselli et al. 2004;Small et al. 2009;Sá Filho et al. 2010).The objective of this experiment was to evaluate P/ AI and follicular growth in response to an injection of 20 mg of FSH or 150 IU of hCG at the time of intravaginal P 4 device removal on a 7-d Co-synch + CIDR protocol in crossbred beef heifers.In this experiment, there was no difference in follicular growth among treatments.No difference was found in the preovulatory follicle diameter at AI day (12 hours after estrus detection or TAI on day 10).Also, no difference was detected among treatments in P/AI or P/AI + NS.However, heifers in the hCG group presented premature ovulation in a greater proportion when compared to the control group and the estrus response was less intense in the hCG group when compared to the control and FSH group.Administration of FSH or hCG at P 4 device removal did not influence follicle growth.A similar result was reported by Sales et al. 2011 when they used 10 mg of FSH or 300 IU of eCG at the time of the P 4 device removal in an estradiol-based protocol.They found a greater preovulatory follicle diameter in the eCG group (13.9 mm) when compared with the FSH group (12.8 mm) or control (12.9 mm) group.In the same study, eCG promoted an increase in follicular growth from day 8 to day 10, which was greater than the FSH and the control groups (eCG: 1.40 mm/day; FSH: 0.90 mm/day; Control: 0.95 mm/day).However, in the study conducted by Souza et al. 2019, a single injection of 150 IU of hCG increased the follicular growth when measured 24 h following the device removal (hCG = 2.04 mm; Control = 1.60 mm).Lack of effect of treatments in follicle growth in the present study was unexpected.
In this experiment, the follicle diameter on day 7 in the FSH and hCG groups was larger than 7 mm.It has been established that the deviation process occurs in follicles > 7 mm, meaning that follicles become LH dependent instead of FSH dependent (Mihm et al. 2006;Acosta et al. 2018).Therefore, the follicle diameter at day 7 could explain one reason why exogenous FSH did not have an effect.On the other hand, the 150 IU of hCG dosage might have induced ovulation instead of helping follicular growth.However, further research should be conducted to identify if this was the case.
Administration of FSH or hCG at intravaginal P4 device removal did not influence P/AI in beef heifers.There are no reports of using either hormone in a GnRH-based protocol at the time of the intravaginal P 4 device removal, as most studies have been conducted with estradiol benzoate-based protocols.Souza et al. 2019 evaluated the use of 150 IU of hCG at the time of removal of the intravaginal P 4 device when using an estradiol-based protocol.In that study, Bos indicus cows were synchronized by applying 2 mg of benzoate estradiol + intravaginal P 4 device containing 750 mg of P 4 at Day 0, one injection of PGF, and intravaginal P 4 device removal at day 8.5 and TAI 36 to 40 hours after device removal.On day 8.5, animals received an injection of 150 IU of hCG (hCG group) or 300 IU of eCG (eCG group) or nothing (control group).The P/AI was greater in the eCG group (53.8%) and hCG group (53.2%) when compared with the control group (43.3%).Regarding the use of FSH, Santos et al. 2007 used an estradiol-based protocol and found an increase in the P/AI when an injection of 400 IU of eCG (60.2% P/AI), 10 mg of FSH (59.7% P/AI), or 20 mg of FSH (58.5% P/AI) was given at the time of the intravaginal P 4 device removal when comparing with the control group (29.9%P/AI).In contrast, Sales et al. 2011 used an estradiol-based protocol in Nellore cows to evaluate the effect of FSH or eCG at P4 device withdrawal.They applied 10 mg of FSH or 300 IU of eCG and there no difference in P/AI between the FSH (60% P/AI) and the control groups (61.5% P/AI) were detected.However, P/AI was greater in the eCG group (71.6%).Exogenous gonadotropins have been used to stimulate the growth of dominant follicles in Bos Indicus cattle, improving P/AI results.However, the benefits of using this strategy have been mostly in animals presenting anestrous, animals with limited feed intake, low body condition score, or in lactating cows (Sa Filho et al. 2009;Sá Filho et al. 2010).The heifers in the current study had on average a BCS of 5 on a scale from 1 to 9; also, 90% were cycling at the beginning of the experiment.Thus, the heifers' physiological stage could help explain why there was no increase in follicle size or follicular growth in this experiment, which resulted in no effect in P/ AI.
Premature ovulation was observed in the present study when hCG was injected at intravaginal P 4 device removal.Among the animals that ovulated prematurely, 17% were from the hCG group.This could be one reason why P/AI did not increase with the application of 150 IU of hCG, as a proportion of animals were inseminated after ovulation.On the other hand, it can be suggested that there was a reduction in the proestrus phase after premature ovulation.Studies have shown that by reducing the proestrus phase, the P/AI is reduced.(Bridges et al. 2010) evaluated the influence of the proestrus length on fertility in lactating and non-lactating beef cows.The proestrus length was managed by changing the PGF-GnRH interval generating two experimental groups: long proestrus (2.25 days of proestrus length) and short proestrus (1.25 days of proestrus length).They found that the P/AI was greater in the long proestrus group (50% of P/AI) than in the short proestrus group (2.6% of P/AI).In the current study, the preovulatory follicle diameter in heifers that presented premature ovulation is unknown; however, there is a possibility that those heifers ovulated smaller or immature follicles (< 10 mm follicles) or they went under atresia.A study reported that animals that ovulated immature follicles had decreased P 4 concentrations during the early luteal phase, and fertility was reduced when immature follicles were induced to ovulate (Mussard et al. 2007).Studies have shown that animals that ovulated smaller follicles had a reduction in fertility compared with those with larger follicles (Pugliesi et al. 2016).Also, the ovulation of small follicles has been related to inappropriate oviductal (Gonella-Diaza et al. 2018, 2015) and uterine environments (Mesquita et al. 2014), resulting in lower embryo receptivity and pregnancy failure.
In the present study, the hCG group expressed a lower estrus response when compared with the other two treatments.This could be explained by the increase in premature ovulations in this group.It has been shown that by decreasing the proestrus phase, there is a reduction in estradiol levels.Bridges et al. 2010 showed that estradiol concentration at estrus was greater in the long proestrus group than in the short proestrus group (12.7 pg/ml vs. 9.1 pg/ml, respectively).This could suggest that animals presenting premature ovulation in the present study also had shorter proestrus and possibly lower estradiol concentrations.This could explain the lower estrus response in the hCG group.
In conclusion, the results in this experiment did not support the original hypothesis.Follicular growth and P/AI were not increased when either a low dosage of FSH or hCG were injected at the time of intravaginal P 4 device removal when using a 7-d Co-Synch + CIDR protocol.Furthermore, 150 IU of hCG increased the number of heifers that underwent early ovulation and decreased the estrus response.Therefore, further research should be conducted to understand follicular dynamics using these two exogenous gonadotropins.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 1 .
Figure 1 .Experimental design and treatments conducted in the present study.A Split-Time AI program was conducted based on the 7 d Co-synch + CIDR protocol.Treatments were applied at the time of intravaginal P 4 device removal (FSH group: 20 mg of FSH; hCG group: 150 IU of hCG; Control: no additional treatment).Heifers that presented estrus 36 hours after intravaginal P 4 device (CIDR) removal were inseminated 12 hours later.Heifers that presented estrus after 36 hours and those that did not present signs of estrus were TAI 60 hours after CIDR removal.

Table 1 .
Effects of treatment (Control, FSH, or hCG) on estrus response, P/AI, P/AI + NS, proportion of premature ovulations, and follicle diameters at day 7 and AI day.
3NS + P/AI = Proportion of animals pregnant by natural service and P/AI 130 days after AI.4Premature ovulations = animals were considered to present premature ovulation if they had a large follicle at P 4 device removal but did not have a follicle of compatible size at the time of artificial insemination.5Folliclediameter of the largest follicle measured at day 7 of the estrus synchronization protocol, the same day of P 4 device withdrawal and administration of treatments.6Folliclediameter of the preovulatory follicle, measured simultaneously with the artificial insemination.

Table 2 .
Relationship between AI day (12 hours after estrus detection or TAI on day 10) and estrus response, P/AI, P/AI + NS, and follicle diameters at day 7 and AI day.
a,bMeans within a row with different superscripts are different (P < 0.05). 1 P/AI = pregnancy per artificial insemination.Thirty days post AI. 2 NS + P/AI = Proportion of animals pregnant by natural service and P/AI 130 days after AI.

Table 3 .
Relationship between pubertal status (Cyclic, Non-Cyclic) and estrus response, P/AI, P/AI + NS, and follicle diameters at day 7 and AI day.P/AI = Proportion of animals pregnant by natural service and P/AI 130 days after AI.