Practical application of an impractical bovine genotype: creating bilateral twin pregnancies in Trio allele carriers

Abstract

Bovine twin birth is associated with detriments, including increased embryo/fetal losses, malpresentation, and dystocia. Incidence of these is lessened in bilateral compared with unilateral twin pregnancy. This study was undertaken to assess the use of follicular ablation by aspiration to create bilateral twin pregnancies in females with genetic potential for ~3.5 ovulations per cycle (Trio allele carriers). In experiment 1, carriers (n = 30) and noncarriers (n = 10) were synchronized for ovulation and timed artificial insemination (TAI). Follicles (>5 mm) in excess of one per ovary were aspirated ~16 h preceding TAI. Follicle count for females with follicles on only one ovary was reduced to two. Blood was sampled 2 wk post-TAI to assess progesterone (P4) concentrations; embryo count was determined by ultrasound 6 wk post-TAI. Circulating P4 concentration post-TAI was significantly (P < 0.001) associated with both genotype and subsequent pregnancy status (pregnant noncarriers: 7.06 ± 0.68 ng/mL; pregnant carriers: 5.54 ± 0.55 ng/mL; nonpregnant noncarriers: 5.22 ± 1.05 ng/mL; nonpregnant carriers: 3.13 ± 0.42 ng/mL). Experiment 2 was undertaken to offset the negative effects of follicular aspiration on subsequent P4 concentration observed in experiment 1. Carriers (n = 38) and noncarriers (n = 32) were submitted to TAI and follicle ablation as described for experiment 1. Additionally, accessory corpora lutea (CL) were induced in carriers by the administration of human chorionic gonadotropin (carriers) at day 6 post-TAI. Consequently, P4 concentration post-TAI was significantly (P < 0.05) associated with subsequent pregnancy status (pregnant: 8.48 ± 0.61 ng/mL; nonpregnant: 6.70 ± 0.63 ng/mL) but not with genotype (carrier: 8.01 ± 0.59 ng/mL; noncarrier: 7.17 ± 0.64 ng/mL). Embryo number was greater in carriers (exp. 1: 1.64 ± 0.81; exp 2: 1.45 ± 0.09) vs. noncarriers (1.00 ± 0.00, both experiments). Single, twin, and triplet pregnancies occurred in carriers in experiment 1, whereas multiples in experiment 2 were limited to twin pregnancies. Genotype effects on pregnancy rate were not significant (P > 0.10) in either experiment. Results suggest that follicular ablation to create bilateral twin pregnancies in Trio carriers is feasible but requires the induction of accessory CL to offset the negative effects of follicular aspiration on subsequent P4 concentration and associated fertility outcomes.

Introduction

Twin pregnancy, while potentially a way to increase the production efficiency in beef cattle, is associated with well-documented detrimental issues, including increased incidence of embryo loss, abortion, malpresentation, dystocia, perinatal calf loss, retained placenta, longer postpartum interval to rebreeding, and infertility in heifer twins born co-twin to males (i.e., freemartins; Echternkamp and Gregory, 1999a, 1999b; Echternkamp et al., 2007). For twinning to be utilized for the improvement of beef cow production efficiency, these problems, with the exception of freemartinism, need to be overcome or lessened. In a beef cattle scenario, the occurrence of freemartin females is insufficient to impact the production of adequate numbers of replacement females and is, therefore, a nonissue.

In a long-running study of twinning in cattle at the USDA Meat Animal Research Center (USMARC), research characterized not only the incidence of the problems mentioned above but also their incidence relative to various types of multiple births or pregnancies, specifically unilateral vs. bilateral twins and triplets. Results from this work indicated greater maintenance of pregnancy, reduced dystocia, and greater calf survival for bilateral twin pregnancies vs. unilateral twin or triplet pregnancies (Echternkamp and Gregory, 1999a, 1999b; Echternkamp et al., 2007). Accordingly, one way of reducing problems associated with twin pregnancy would be to specifically produce bilateral twin pregnancies, while avoiding the production of unilateral twin pregnancies and higher-order multiple births, as has been done previously using embryo transfer (Gordon and Boland, 1979; Anderson et al., 1982; Sreenan and Diskin, 1989).

Our recent work has characterized a single gene with a profound effect on bovine ovulation rate, referred to as the Trio allele (Kirkpatrick and Morris, 2015). The Trio allele in heterozygous (carrier) females causes the ovulation rate to increase from the typical single dominant follicle and ovulation to an average of 3.5 ovulations per cycle (García-Guerra et al., 2017b). Trio carriers have similar circulating progesterone (P4) as noncarriers corresponding to a similar volume of luteal tissue, although this is contained in multiple corpora lutea (CL) in carriers and only one CL in noncarriers (García-Guerra et al., 2018b). The occurrence of multiple dominant follicles in these females, often bilaterally, suggests the possibility that they could be utilized to produce bilateral twin pregnancies by selective ablation of dominant follicles in excess of one on each ovary by follicular aspiration. Thus, the current study was conducted to test the viability of using the Trio allele carrier genotype and selective follicular aspiration to produce bilateral twin pregnancies, in comparison to normal, noncarrier, half-sib females.

Materials and Methods

The protocols used in this study were approved by the College of Agricultural and Life Sciences Animal Care and Use Committee at the University of Wisconsin-Madison (Madison, WI, USA).

Experiment 1: animals and design

Animals used were a mix of nulliparous, primiparous, and multiparous females. The noncarrier group consisted of 10 multiparous females ranging from 3 to 11 yr of age. The carrier group was comprised of 20 nulliparous, 9 primiparous, and 1 multiparous female with ages of 3 and 4 yr. Trio allele genotype (carrier vs. noncarrier) status was determined by haplotyping as previously described (Kirkpatrick and Morris, 2015). All females were of primarily Angus breeding; in two cases, noncarriers were paternal half-sibs to carriers, while the remainder of noncarriers were unrelated Angus-cross females.

A modified 5-d CO-synch plus controlled internal drug release device (CIDR) protocol was used to synchronize estrus (Figure 1), consisting of 100 µg gonadotropin-releasing hormone (GnRH; gonadorelin hydrochloride, Factrel, Zoetis Inc., Parsippany, NJ) and P4 pessary containing 1.38 g P4 (Eazi-Breed CIDR, Zoetis, Parsippany, NJ) application on day 8, followed by CIDR removal and administration of two consecutive 5 mL doses (25 mg dinoprost each) of prostaglandin F_2-alpha (Lutalyse, Zoetis, Parsippany, NJ) 8 h apart on day 3 relative to timed artificial insemination (TAI) (day 0). TAI was performed 66 h following CIDR removal, and GnRH was administered immediately following TAI. Synchronization and artificial insemination (AI) of noncarrier and carrier females took place during the weeks of July 22nd and 29th, 2019, respectively. All females were bred to the same AI sire.

Figure 1.

Design of experiments 1 and 2. Notable changes in the design of experiment 2 are the addition of a pre-synchronization (prostaglandin at day 18 and GnRH at day 15) prior to the synchronization leading to TAI, administration of a double dose of GnRH at the time of administration of the CIDR, and administration of hCG (day 6) to induce the formation of accessory CL.

Experiment 2: animals and design

Animals used consisted of nulliparous females with ages of approximately 18 or 30 mo at the start of the trial. Animals were grouped by age with the groups beginning the experiment in two successive weeks in November 2019. Breed make-up and determination of carrier status are as described above for experiment 1 with the difference that most carriers and noncarriers within age groups were paternal half-sibs.

A substantially modified protocol was used in experiment 2 (Figure 1). Changes to the protocol included pre-synchronization with prostaglandin 9 d (day 18) and GnRH 6 d (day 15) prior to the administration of a P4 pessary, a double dose of GnRH (200 µg) to stimulate a greater luteinizing hormone peak, as is needed to stimulate the ovulation in the presence of higher circulating P4 and lower estradiol (day 9; Giordano et al., 2012, 2013), inclusion of prostaglandin F2-alpha (PGF) at the time of CIDR insertion to reduce circulating P4 and potentially increase follicular growth during the protocol (Wiltbank et al., 2014), treatment with two doses of PGF on days 3 and 2 (24 h apart) to assure regression of the CL (Nascimento et al., 2014), a reduction in the duration between CIDR removal and TAI to 48 h since the first PGF treatment was given on day 3, and administration of GnRH at the time of follicular aspiration. Administration of human chorionic gonadotropin (hCG) on day 6 post-TAI was added to the protocol for the induction of accessory CL. Blood samples were obtained, as described above, on days 6 and 13 following TAI and weekly starting on day 28 and ending on day 55 following TAI for the determination of P4 and pregnancy-specific protein B (PSPB) concentrations.

GnRH was administered at the time of follicular aspiration in both experiments based on the results from previous studies (Pursley et al., 1998; Brusveen et al., 2008) indicating the optimal time to induce ovulation is around 16 h before AI. Thus, since ovulation occurs ~28 h after GnRH administration, this would be ~12 h after AI, producing an optimal time of the oocyte to arrive in the oviduct to be fertilized by the capacitated sperm.

Follicular ablation and ultrasound evaluations

Dominant follicles in Trio allele carriers attain deviation at diameters smaller than in noncarriers (5.5 vs. 8.3 mm; García-Guerra et al., 2018a). Thus, the follicle diameter was the criterion used to determine which follicles were dominant. Dominant follicles in excess of one per ovary were aspirated with the largest follicle being left intact. When carrier females had potentially dominant follicles on only one ovary, follicles in excess of two were aspirated. Follicular aspiration was performed using transvaginal ultrasound to visualize the ovary and aspiration needle. Aspiration took place ~16 h prior to TAI in carrier females. Noncarrier females were not examined by ultrasound before AI, and none received follicular aspiration.

Ultrasound evaluation was used to assess the number of CL, pregnancy status, and embryo count. In experiment 1, count of embryos and CLs took place on the same day for all females at 46 and 39 d of gestation for noncarrier and carrier females, respectively. In experiment 2, the number of CL was determined at days 6 and 13 post-TAI, and pregnancy status and embryo counts were determined weekly from day 28 through day 48.

Assessing P4 and PSPB concentration

Luteal function was evaluated by P4 concentrations in blood samples collected by coccygeal venipuncture and transrectal ultrasonic evaluation of CL on day 12 following TAI in experiment 1 and at days 6 and 13 and weekly from day 21 through day 49 in experiment 2. Blood samples were centrifuged at 1,300 × g at 4 °C for 20 min, and serum transferred into vials, frozen, and stored at –20 °C until assayed. P4 concentration was assayed using a solid-phase radioimmunoassay (RIA) kit containing antibody-coated tubes and 125I-labeled P4 (ImmuChem Coated Tube Progesterone 125I RIA Kit, MP Biomedicals, Costa Mesa, CA, USA). The P4 intra-assay coefficient of variation (CV) and sensitivity were 11.17% and 0.15 ng/mL, respectively, for experiment 1. Intra- and inter-assay CVs for experiment 2 were 5.7% and 8.4%, respectively. The sensitivity for P4 assays in experiment 2 was 0.08 ng/mL. Concentrations of PSPB were determined using a commercial ELISA assay (Biopryn, BioTracking LLC, Moscow, ID) as described previously (Toledo et al., 2017). The PSPB intra- and inter-assay CVs were 4.1% and 10.8%, respectively, with a sensitivity of 0.06 ng/mL.

Statistical analysis

Association of P4 concentration with genotype and pregnancy status was analyzed using analysis of variance in the stats R package (R Development Core Team, 2013) with a model including fixed effects of genotype (carrier vs. noncarrier) and pregnancy status (pregnant vs. nonpregnant) and their interaction in experiment 1. For experiment 2, where P4 was assayed on a weekly basis, data were analyzed with a repeated records model including animal as a random effect. Least-squares means and standard deviation were determined using the emmeans package in R (https://github.com/rvlenth/emmeans). Independence of genotype and pregnancy status was tested by Chi-square test in R. Genotype effects on embryo count were tested by t-test considering the null hypothesis of no effect vs. the one-sided alternative of Trio allele carriers having greater embryo count.

Results

Experiment 1

As anticipated, carrier females typically had multiple follicles of 5 mm or greater at the time of follicular aspiration (3.60 ± 0.30, Table 1). Genotype and pregnancy status had highly significant (P < 0.001) associations with P4 concentration but their interaction was not significant (P > 0.05). Estimated marginal means for P4 were 7.06 ± 0.68, 5.54 ± 0.55, 5.22 ± 1.05, and 3.13 ± 0.42 ng/mL for pregnant noncarriers, pregnant carriers, open noncarriers, and open carriers, respectively. The range of P4 values was nearly twice as great for carriers vs. noncarriers (Figure 2).

Table 1.

Effect of genotype on CL number and diameter and pregnancy rate

Experiment 1	Noncarriers (n = 10)	Carriers (n = 30)
No. of follicles prior to aspiration	NA	3.60 ± 0.30
No. of CL, day 12	1.10 ± 0.36	2.47 ± 0.21**
Pregnancy rate, day 39 to 46 of gestation	0.70 (7/10)	0.37 (11/30)
Embryo count, week 6 of gestation	1.00 ± 0.00	1.64 ± 0.81**
Experiment 2	Noncarriers (n = 32)	Carriers (n = 38)
No. of follicles prior to aspiration	NA	3.37 ± 0.21
No. of CL, day 6	1.03 ± 0.11	2.32 ± 0.10***
No. of CL, day 13	1.09 ± 0.18	4.83 ± 0.17***
Average CL diameter, day 6	19.9 ± 0.53	15.5 ± 0.50***
Pregnancy rate, day 28 of gestation	46.9 (15/32)	57.9 (22/38)
Pregnancy rate, day 34 of gestation	43.8 (14/32)	57.9 (22/38)
Pregnancy rate, day 41 of gestation	40.6 (13/32)	57.9 (22/38)
Pregnancy rate, day 48 of gestation	40.6 (13/32)	52.6 (20/38)
Embryo count, day 41 of gestation	1.00 ± 0.00	1.45 ± 0.09**

**Effect of genotype significant at P < 0.01.

*** Effect of genotype significant at P < 0.001.

Figure 2.

Box plot showing the distribution of progesterone concentration day 12 post breeding by genotype (Trio allele carrier vs. noncarrier) and pregnancy status at day 39 to 46 in experiment 1. Effects of genotype and pregnancy status were highly significant (P < 0.001). The lower and upper ends of boxes correspond to the first and third quartiles, respectively. The horizontal line dividing the box represents the median. Whiskers extend to the smallest and largest values no further than 1.5 times the interquartile range from the first or third quartile.

The number of embryos (Figure 3) was greater (P < 0.05) in carrier than noncarrier females, 1.64 ± 0.81 vs. 1.00 ± 0.00. Embryo counts of 1, 2, and 3 were observed among carrier females, while noncarrier females had only singles (Figure 3). The difference in pregnancy rate at 6 wk of gestation between noncarriers (70%, 7/10) and carriers (37%, 11/30) was not statistically significant.

Figure 3.

Embryo count by dam genotype (Carrier, red; noncarrier, blue) at 6 wk of gestation in experiment 1 (exp1) and experiment 2 (exp2). The average embryo count for each genotype is shown and denoted in the figure by black diamonds. Effect of genotype was significant (P < 0.01).

Experiment 2

As expected, carrier females typically had multiple follicles of 5 mm or greater at the time of follicular aspiration (3.37 ± 0.21, Table 1). The association of genotype with P4 concentration was highly significant 6 d following TAI (carrier 2.68 ± 0.23 ng/mL vs. noncarrier 3.57 ± 0.26 ng/mL; P < 0.01), but no difference (P > 0.05) between genotypes was observed 1 wk later at 13 d post-TAI (Table 1; Figure 4) after the induction of accessory CL in carriers (carrier 8.09 ± 0.61 ng/mL vs. noncarrier 6.96 ± 0.67 ng/mL). Subsequent pregnancy status at day 28 post-TAI was significantly (P < 0.05) associated with circulating P4 concentration at both day 6 (pregnant 3.51 ± 0.25 ng/mL vs. nonpregnant 2.74 ± 0.25 ng/mL) and day 13 post-TAI (pregnant 8.33 ± 0.64 ng/mL vs. nonpregnant 6.72 ± 0.64 ng/mL). Differences in circulating P4 concentrations between age groups were not significant. Effects of genotype on circulating P4 concentrations in pregnant females at day 28 through day 49 post-TAI were not significant, though the effects of gestation day were significant (P < 0.01; Figure 5). Genotypes did differ significantly in variation in P4 concentration, with carrier exhibiting greater variation than noncarriers (σ _carrier = 5.54 ng/mL vs. σ _noncarrier = 2.54 ng/mL; P < 0.01; Figure 5).

Figure 4.

Progesterone concentration (mean and 95% confidence interval) at days 6 and 13 following TAI by genotype and day 28 pregnancy status in experiment 2. Effect of genotype significant (P < 0.01) at day 6 prior to the induction of accessory CL, but not significant (P > 0.10) on day 13 following the induction of accessory CL. Pregnancy status at day 28 of gestation significantly (P < 0.05) associated with P4 at both days 6 and 13.

Figure 5.

Box plot of progesterone concentration at day 21 through day 48 by genotype for females pregnant at day 48. Effect of genotype was significant for variation but not mean of progesterone concentration.

The number of embryos was greater (P < 0.05) in carrier than noncarrier females, 1.45 ± 0.09 vs. 1.00 ± 0.00. Embryo counts of 1 and 2 were observed among carrier females, while noncarrier females had only singles (Figure 3). The difference in pregnancy rate at day 28 through day 49 of gestation between carriers and noncarriers (46.9% to 40.6% for noncarriers and 57.9% to 52.6% for carriers; Table 1) was not statistically significant.

Concentrations of PSPB at day 28 of gestation were associated with female age (18 mo 3.14 ± 0.22 ng/mL vs. 30 mo 2.35 ± 0.21 ng/mL; P < 0.05) and pregnancy type (twins 3.26 ± 0.257 vs. singles 2.23 ± 0.167 ng/mL; P < 0.01) (Figure 6). Main effects of female age on PSPB concentrations at day 34 of gestation (Figure 7) were not significant (P > 0.10), though main effects of pregnancy type (P < 0.001) and interaction of dam age and pregnancy type (P < 0.05) were significant (18 mo with singleton: 3.89 ± 0.46 ng/mL; 18 mo with twins: 8.39 ± 0.89 ng/mL; 30 mo with singleton: 3.65 ± 0.54 ng/mL; 30 mo with twins: 4.66 ± 0.72 ng/mL). Despite the statistically significant effect of pregnancy type at both time points, there was considerable overlap in PSPB concentrations between pregnancy types (Figures 6 and 7).

Figure 6.

Box plot for PSPB on day 28 following TAI by embryo count (single vs. twin) and dam age (18 vs. 30 mo). Effects of embryo count (P < 0.01) and dam age (P < 0.05) were statistically significant. The lower and upper ends of boxes correspond to the first and third quartiles, respectively. The horizontal line dividing the box represents the median. Whiskers extend to the smallest and largest values no further than 1.5 times the interquartile range from the first or third quartile.

Figure 7.

Box plot for PSPB on day 34 following TAI by embryo count (single vs. twin) and dam age (18 vs. 30 mo). Effects of embryo count (P < 0.001) and the interaction of embryo count and dam age (P < 0.05) were statistically significant. The lower and upper ends of boxes correspond to the first and third quartiles, respectively. The horizontal line dividing the box represents the median. Whiskers extend to the smallest and largest values no further than 1.5 times the interquartile range from the first or third quartile.

Discussion

This study was designed to validate a technique to produce bilateral twin pregnancies in beef cattle that are carriers of the Trio allele and consequently have multiple (~3.5) spontaneous ovulations (García-Guerra et al., 2017b). Thus, we used a combination of reproductive techniques, that is, estrous synchronization protocols and follicle aspiration to selectively ablate dominant follicles in excess of one on each ovary. Among the most important findings of experiment 1 is the reduction in P4 concentration in follicular-aspirated carriers vs. noncarriers. Previous work with carrier and noncarrier females has reported similar circulating P4 and similar total luteal volume with multiple, smaller diameter CL in Trio allele carriers compared with a single, larger CL in noncarriers (García-Guerra et al., 2017a; García-Guerra et al., 2018b). The results of this study suggest that the follicular aspiration performed on carriers compromised subsequent P4 production by reducing the number of CL. The reduced P4 concentration at day 12 post-TAI in females subsequently determined to be nonpregnant vs. pregnant suggests that the reduced P4 compromised maintenance of pregnancy. Consequently, to effectively create twin pregnancies in Trio allele carrier females, it may be necessary to induce the development of accessory CL by the administration of GnRH or hCG post breeding (Nascimento et al., 2013; Baez et al., 2017) as was conducted in experiment 2.

The follicular aspiration employed failed to result in consistent production of twin pregnancies in experiment 1. These results raised a question concerning the accuracy of assessment of follicle status based on the simple criterion employed (follicular diameter). The production of singles in more than half of the pregnant carriers suggests the possibility that the follicles chosen to remain intact may have in some cases been atretic follicles not capable of ovulating a viable oocyte. In contrast, the occurrence of three embryos in two cases suggests that follicles below 5 mm in diameter which were not aspirated had the potential to ovulate subsequently in some cases. An improved criterion or set of criteria for determining follicle status regarding dominance might improve the consistency of production of twin pregnancies, though it is not clear what criteria could be as quickly and simply assessed as follicle diameter.

Results from experiment 2 provide strong evidence that accessory CL could be produced and circulating P4 concentration increased to concentrations comparable as observed in noncarrier females. Decreased P4 concentrations in carriers vs. noncarriers on day 6 following TAI reflected the reduction in CL number caused by follicular aspiration prior to TAI, while the increase in CL count and comparable P4 concentrations at day 13 post-TAI indicates that the induction of accessory CL increases circulating P4, similar to previous studies (Thatcher et al., 2001; Rizos et al., 2012; García-Guerra et al., 2020). Previous studies have also indicated that increasing circulating P4 by the induction of an accessory CL can increase fertility following AI or reduce pregnancy loss after embryo transfer (Niles et al., 2019; García-Guerra et al., 2020). Also, the modifications to the protocol, adding a pre-synchronization induction of a follicular wave and luteal regression (Silva et al., 2007; Dewey et al., 2010; Mendonça et al., 2019), likely provided greater synchrony of folliculogenesis and more effective selection of follicles for ablation, as only single and twin pregnancies were produced (no triplets, as in experiment 1). The reduction in interval from CIDR removal to follicular aspiration and TAI may have also helped in improving conception for animals ovulating earlier.

Significant differences between single and twin gestations were observed for PSPB at both 28 and 34 d of gestation; however, there was considerable overlap in circulating PSPB concentration for twins and singles. However, it seems doubtful that PSPB concentration, alone, could be used effectively as an indicator of embryo number. Previous studies have similarly found significant differences in PSPB (Dobson et al., 1993; Patel et al., 1995; Szelényi et al., 2018) and pregnancy-associated glycoprotein-1 (Schmidt et al., 1996; Patel et al., 1997; Serrano et al., 2009) between gestation types, but not of sufficient magnitude for the accurate prediction of embryo number.

Results from this study provide a proof of concept that the creation of bilateral twin pregnancies by employing selective follicular ablation in a high ovulation rate genotype is technically feasible (9 of 10 twin pregnancies at day 41 of gestation were bilateral). In addition, transuterine migration of embryos appears to be rare in cattle based on the observation that all pregnancies were found to be ipsilateral to the CL in two large studies in cattle (Scanlon, 1972; Borges et al., 2017) and consistent with our observation in this study. An advantage of the genotype used here is that it is due to a single gene that confers a high ovulation rate in heterozygotes (carriers). Therefore, the introduction of the allele and creation of females with potential for high ovulation rate could occur in one generation in a beef cattle herd. In the context of a rotational mating system in beef cattle, the allele could be introduced initially by mating females to Trio allele carrier or homozygote males to produce carrier females, then subsequently genotyping female offspring from carrier dams to identify carrier daughters to retain as replacement females; the introduction of the allele into the herd would occur only at the initial generation.

An alternative application of the Trio allele genotype is worth consideration in the context of production of bilateral twin pregnancies. The frequent production of CL on both ovaries in Trio allele carriers suggests the possibility that they might serve as ideal recipients for the bilateral transfer of embryos. It is well documented (Tervit et al., 1977; Brand and Akabwai, 1978; Del Campo et al., 1983) that embryo survival is far greater for transfer to the ipsilateral vs. contralateral uterine horn with regard to the presence of a CL in females with one CL. Trio allele carriers, in contrast, commonly have no contralateral uterine horn. Suitability of Trio allele carriers as recipients for bilateral embryo transfer remains to be evaluated.

Glossary

Abbreviations

AI

artificial insemination

CIDR

controlled internal drug release device

CL

corpus luteum

GnRH

gonadotropin-releasing hormone

hCG

human chorionic gonadotropin

P4

progesterone

PSPB

pregnancy-specific protein B

TAI

timed artificial insemination

Conflict of interest statement

All authors have no conflicts of interest.