The effect of clitoral stimulation post artificial insemination on pregnancy rates of multiparous Bos indicus beef cows submitted to estradiol/progesterone-based estrus synchronization protocol (2024)

Abstract

Clitoral stimulation after artificial insemination (AI) in beef cattle is a common practice utilized by AI technicians; however, the effect of clitoral stimulation during fixed-time AI (TAI) is still unknown. The objective of the present experiment was to evaluate the effect of clitoral stimulation on temperament responses and pregnancy rates to TAI in Bos indicus beef cows. A total of 1,186 multiparous Nellore cows across three different locations were assigned to an estradiol/progesterone-based estrus synchronization + TAI protocol (day −11 to 0). Cows were randomly assigned to receive either 3 s of clitoral stimulation (n = 602) or no clitoral stimulation (n = 584) immediately after TAI (day 0). Cow body condition score (BCS) was recorded on day −11. Estrus expression was evaluated based on estrus detection visual aid patch activation on day 0 (estrus, ≥50% activated; no estrus, <50% activated). Temperament was assessed by individual chute score based on a 5-point scale before TAI, and individual exit velocity was measured after clitoral stimulation. Pregnancy diagnosis was performed 30 d after TAI via transrectal ultrasonography. Pregnancy rate to TAI was positively affected by BCS (P < 0.01) and estrus expression (P = 0.03). Pregnancy rates of cows receiving clitoral stimulation did not differ (P = 0.39) from cows non-stimulated (47.5 ± 4.6% vs. 44.3 ± 4.6%, respectively). No interaction of clitoral stimulation and estrus expression was observed (P = 0.26). Chute score was positively correlated to exit velocity (P < 0.01; r = 0.29); however, clitoral stimulation did not affect exit velocity (P = 0.86). In summary, pregnancy rates to TAI are influenced by various factors and multiple strategies have the potential to increase the fertility of beef cows submitted to TAI; however, clitoral stimulation of Bos indicus beef cows did not improve TAI pregnancy rate.

Keywords: beef cows, clitoral stimulation, fixed-time artificial insemination

Introduction

Artificial insemination (AI) has been used for many years and allow cattle producers to use superior sires and improve the genetic merit of the beef herd; however, the adoption of this technology is still limited, especially due to the reduced pregnancy rates and increased animal handling and requirement of estrus detection. Considerable efforts have been devoted to developing technologies and strategies to improve reproductive performance, as well as the overall cattle productivity to make AI more attractive to cattle producers.

Previous research reported that manual clitoral stimulation for few seconds after AI increased the pregnancy rate in beef cows (Randel et al., 1975; Short et al., 1979; Lunstra et al., 1983). Other studies in dairy cows and beef heifers, however, failed to demonstrate any beneficial effect of clitoral stimulation on pregnancy rates to AI (Randel et al., 1975; Lunstra et al., 1983; Cooper et al., 1985). Clitoral stimulation has been reported to cause a single immediate uterine contraction (Cooper et al., 1985), hasten the luteinizing hormone (LH) surge, and shorten the interval from the beginning of estrus to ovulation in cows (Randel et al., 1973). Moreover, clitoral stimulation is suggested to have a neural response rather than hormonal response due to the sensory receptors in the clitoral region and neural pathways to the uterus (Cooper and Foote, 1986). Increased excitability has also been observed after clitoral stimulation in heifers (Cooper et al., 1985); however, the physiological reasons for the differing responses of dairy cattle, beef cows, and beef heifers remain unclear. Nevertheless, clitoral stimulation is an extensively used technique practiced by most field AI technicians in beef operations.

Hormonal treatments have been developed to control follicular wave dynamics and time of ovulation, allowing fixed-time AI (TAI), without the need of estrus detection. The adoption of TAI protocols has the potential to shorten the calving season, increase calf uniformity, and facilitate the use of AI. Incorporation of progesterone, estradiol, prostaglandin F2α (PGF), and equine chorionic gonadotrophin (eCG) has been reported to be efficient in stimulating the final development of the dominant follicle and induce ovulation in Bos indicus beef cattle, achieving satisfactory ovulation rates and pregnancy rates (Sá Filho et al., 2009; de Sá Filho and Vasconcelos, 2011). Several factors have been reported to influence pregnancy rates to TAI (Vasconcelos et al., 2017); however, no study has investigated the effect of clitoral stimulation in pregnancy rates of beef cows submitted to TAI. Therefore, the objective of the current study was to evaluate the effect of clitoral stimulation on temperament responses and pregnancy rates of Bos indicus beef cattle submitted to an estradiol/progesterone-based TAI protocol. We hypothesized that clitoral stimulation does not affect the pregnancy rates and can potentially induce excitable temperament in beef cows submitted to TAI.

Materials and Methods

Animals and reproductive management

All cows were managed following the recommendations of the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 2010). A total of 1,186 postpartum multiparous Nellore cows from 14 different groups across 3 different locations were assigned to the experiment. Groups were allocated according to the general scheme of the operation with approximately 85 cows in each group (range = 55 to 122). All animals enrolled in the experiment were maintained on pastures, specifically Brachiaria brizantha with ad libitum access to water and mineral–vitamin supplement.

Cows were enrolled to the following estrus synchronization protocol: an intravaginal progesterone (P4) insert containing 1.9 g of P4 (CIDR, Zoetis, Sao Paulo, Brazil) and 2.0 mg (i.m.) of estradiol benzoate (2.0 mL of Gonadiol; Zoetis, Sao Paulo, Brazil) on day −11, 12.5 mg (i.m) of dinoprost tromethamine (PGF; 2.5 mL of Lutalyse; Zoetis, Sao Paulo, Brazil) on day −4, CIDR withdrawal, 300 IU (i.m.) of eCG (1.5 mL of Novormon; Zoetis, Sao Paulo, Brazil), and 0.6 mg (i.m.) of estradiol cypionate (0.3 mL of E.C.P.; Zoetis, Sao Paulo, Brazil) on day −2, and TAI on day 0. Estrotect Breeding Indicators (Estrotect; Rockway Inc., Spring Valley, WI) were placed on day −2 and scored on day 0 on a scale from 0 to 4 (0, missing patch; 1, <25% activated; 2, 25% to 50% activated; 3, 50% to 75% activated; 4, >75% activated; Pohler et al., 2016; Franco et al., 2018). Cows with a patch score of 0 were not included in the analyses; cows with patch score 1 or 2 were classified as not expressing estrus; and cows with patch scores 3 and 4 were classified as expressing estrus. On day −11, body condition score (BCS) was determined on a scale from 1 to 9 (Wagner et al., 1988). All cows were diagnosed for pregnancy by transrectal ultrasonography (5.0 MHz transducer; 500 V, Aloka, Wallingford, CT) 30 d after TAI, based on the presence of a viable embryo (detection of a heartbeat).

Experimental procedure

Cows were inseminated by 7 experienced technicians with semen from 10 randomized Angus sires of proven fertility. Immediately following TAI, cows were randomly assigned to receive either no clitoral stimulation (n = 584) or 3 s of manual clitoral stimulation (n = 602). Immediately before TAI, individual chute score was assessed by a single technician based on a 5-point subjective scale (1 = calm with no movement; 2 = restless movements; 3 = frequent movement with vocalization; 4 = constant movement, vocalization, and shaking of the chute; and 5 = violent and continuous struggling) and classified according to the chute score as adequate temperament (chute score ≤ 3) or excitable (chute score > 3) based on Cooke (2014). Individual exit velocity was assessed immediately after AI by determining the rate of travel over a 2.0-m distance upon exiting the chute with an infrared sensor (FarmTek Inc., North Wylie, TX).

Statistical analysis

All data were analyzed using cow as the experimental unit and the Satterthwaite approximation to determine the denominator df for the tests of fixed effects. Quantitative data (BCS, exit velocity, and chute score) were analyzed with the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC) with cow (treatment × group) as a random variable. Binary data (pregnancy rates and estrus expression) were analyzed with the GLIMMIX procedure of SAS with cow (treatment × group) as a random variable for estrus expression and cow (treatment × group), with the addition of sire and AI technician as random variables for the analysis of pregnancy rates. All model statements contained the effects of treatment (clitoral stimulation or not), in addition to estrus expression and its interaction with treatment for pregnancy analysis, and chute score as an independent covariate for exit velocity analysis. For all analyses, significance was set at P ≤ 0.05.

Results

BCS average was equal (BCS = 5.03 ± 0.01) in both treatments, and no effect (P = 0.16) of BCS was detected for cows either receiving or not receiving clitoral stimulation. The incidence of cows expressing estrus at TAI was 52.4% and also did not differ (P = 0.28) between treatments. Hence, any reproductive difference between treatments should not be attributed to BCS or estrus expression. Estrus expression and pregnancy rates to TAI were affected by BCS, where cows presenting greater BCS at the beginning of the estrus synchronization protocol had greater (P < 0.01) estrus expression and greater (P = 0.03) pregnancy rate. Pregnancy rate to TAI was affected by estrus expression, where cows classified as expressing estrus at TAI had greater (P < 0.01) pregnancy rates when compared with cows classified as not expressing estrus (60.1 ± 4.6% vs. 37.6 ± 4.6%, respectively).

Manual clitoral stimulation for 3 s at TAI did not affect pregnancy rate to TAI (P = 0.39) when compared with cows not receiving clitoral stimulation (47.5 ± 4.6% vs. 44.3 ± 4.6%, respectively; Figure 1), and no interaction of clitoral stimulation by estrus expression was observed (P = 0.26). Pregnancy rate to TAI of cows not expressing estrus did not differ (P = 0.82) between cows receiving clitoral stimulation or no clitoral stimulation (33.2 ± 5.1% vs. 34.1 ± 5.0%, respectively). Similarly, pregnancy rates to TAI did not differ (P = 0.16) in cows that expressed estrus and received clitoral stimulation or no clitoral stimulation (59.7 ± 4.9% vs. 54.2 ± 5.0, respectively; Figure 1).

Figure 1.

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Exit velocity and chute score were positively correlated (r = 0.29). Cows considered excitable (chute score > 3) had increased (P < 0.05) exit velocity compared with cows with adequate temperament (chute score ≤ 3); however, clitoral stimulation following TAI did not alter the exit velocity of the cows regardless their chute score before TAI (P = 0.86; Figure 2).

Figure 2.

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Discussion

Previous studies have reported that short periods of clitoral stimulation immediately after AI increase pregnancy rates of beef cows. A few decades ago, in an experiment with more than 1,000 beef females, Randel et al. (1975) reported an increase of 6.3% on the conception rate of cows receiving 10 s of clitoral stimulation. Similarly, Segura and Rodriquez (1994) reported an increase of 12% in pregnancy rates when clitoral stimulation was applied 10 times following AI to Bos indicus crossed heifers. Lunstra et al. (1985) and Short et al. (1979) demonstrated that 3 s of clitoral stimulation immediately after AI was sufficient to increase the pregnancy rates in Bos taurus cows. It is important to point out that these research studies were conducted using clitoral stimulation after AI following estrus detection and not TAI. In the present study, clitoral stimulation for 3 s after TAI did not increase the pregnancy rate in Bos indicus beef cows submitted to an estradiol-based estrus synchronization protocol. Similar results were also observed in dairy cows stimulated for 5 s (Cooper et al., 1985) and heifers stimulated from 3 to 10 s following AI (Randel et al., 1975; Lunstra et al., 1983, 1985). The physiological response to the stimulation of the bovine clitoris is still unknown; however, it seems that the uterine contraction (Cooper et al., 1985) and the hastening of the LH surge and ovulation (Randel et al., 1973), reported to be responsible for the increase in pregnancy rates, do not affect the pregnancy rates of Bos indicus beef cows submitted to a TAI protocol.

Neurological pathways are reported to exist between the ovine reproductive tract and the pituitary gland (Robertson and Rakha, 1965). According to Cooper et al. (1985), 30 s of clitoral stimulation caused a single uterine contraction but no effects on oxytocin concentration were observed, suggesting a neural-mediated response. The same authors also reported that heifers receiving clitoral stimulation tended to exhibit nervousness, suggesting a change in behavior due to the clitoral stimulation. No other studies, however, have investigated the temperament responses of cows to clitoral stimulation. In the present study, clitoral stimulation did not affect the exit velocity of the cows, suggesting that the neural response of the manual clitoral stimulation does not affect the behavior of the cows, independent of individual temperament. Accordingly, temperament of B. indicus beef cows has been directly associated with their reproductive performance, as cows classified with adequate temperament having greater pregnancy rates to TAI compared with cows classified with excitable temperament (Cooke et al., 2011, 2017).

Several researches have reported different factors affecting the reproductive performance of cows submitted to TAI protocols, including nutritional management (Perry, 2016), hormonal treatments (Baruselli et al., 2004; Sa Filho et al., 2009), genetic selection (Berry et al., 2014), animal handling (Cooke et al., 2017), insemination technique, and semen handling (Diskin and Kenny, 2016). The pharmacological synchronization of estrus with progesterone and estradiol suppresses follicle-stimulating hormone and follicular growth, and synchronize follicular wave emergence in bovine females regardless of the stage of follicular development at the time of treatment (Bo et al., 1994). The use of ovulatory stimulus estradiol cypionate (ECP) and gonadotropic support (eCG) 48 h prior to TAI has been reported to achieve satisfactory ovulation rates (~85%) and pregnancy rates (~50%) in Bos indicus beef cows (Sá Filho et al., 2009, 2010). One important consideration is that these types of protocols, using ECP and eCG, that support the final maturation of the developing follicle and induce ovulation may limit the impact of clitoral stimulation on pregnancy rates as reported previously in the literature. Estrus expression at TAI and preovulatory estradiol concentration are directly correlated with pregnancy success in beef and dairy cows (Perry et al., 2005; Pohler et al., 2012; Pereira et al., 2016). In addition, Perry et al. (2005, 2007) reported that beef heifers and cows exhibiting estrus within 24 h of TAI have greater fertility compared with cows that do not exhibit estrus. Similar outcomes were observed in the present study, with cows standing in estrus prior to TAI having 23% greater pregnancy rates compared with cows not expressing estrus. Therefore, critical factors such as sire/semen quality, AI technician, nutrition, and reproductive management have a greater effect on reproductive performance than any individual component of the insemination process such as clitoral stimulation.

Acknowledgments

We would like to thank United States Department of Agriculture - National Institute of Food and Agriculture Hatch/Multistate Project W3112-TEX07702—Reproductive performance in domestic animals for financial support and ESTROTECT, Inc. (Spring Valley, WI) for their donation of Estrotect heat detector patches used in the experiment.

Glossary

Abbreviations

AI: artificial insemination
BCS: body condition score
eCG: equine chorionic gonadotrophin
ECP: estradiol cypionate
FSH: follicle-stimulating hormone
LH: luteinizing hormone
P4: progesterone
PGF: prostaglandin F2α
TAI: fixed-time artificial insemination

Conflicts of interest statement

The authors declare no actual or potential conflicts of interest that affect their ability to objectively present or review research or data.

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