J. Anim. Sci. 2003. 81:821-829
© 2003 American Society of Animal Science
Influence of hormone supplementation to extended semen on artificial insemination, uterine contractions, establishment of a sperm reservoir, and fertility in swine1,2
K. L. Willenburg,
G. M. Miller,
S. L. Rodriguez-Zas and
R. V. Knox3
Department of Animal Sciences, University of Illinois, Urbana 61801
3 Correspondence:
360 Animal Sciences Laboratory, 1207 W. Gregory Dr. (phone: 217-244-5177; fax: 217-333-8286; E-mail:
rknox{at}uiuc.edu).
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Abstract
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This study was performed to quantify the effect of hormone addition to semen using a low-fertility model to evaluate its effectiveness and mode of action. At 24 h after the onset of estrus, all gilts received a single low-dose AI (0.5 x 109 sperm/80 mL) with no hormone (control, C), estrogens (E, 11.5 µg), PGF2
(PG, 5 mg of Lutalyse), or oxytocin (OT, 4 IU), which were then evaluated for semen backflow (n = 48), oviductal and uterine sperm numbers (n = 28), uterine contractions (n = 12), pregnancy rate (PR, n = 120), and number of fetuses (n = 67). In Exp. 1, backflow of semen from the uterus was collected for 8 h after AI, whereas PR and fetuses were assessed at d 25 to 30 after AI. In Exp. 2, backflow was collected and reproductive tracts flushed to determine sperm numbers in the oviducts and the anterior segments of the uterus. In Exp. 3, sows were monitored for uterine contractions for 1 h before AI and for 2 h after AI. In Exp. 1, there was a treatment x time interaction for fluid loss (P < 0.001), but by 8 h after AI, there was no difference in the total volume (70 ± 1 mL) of semen lost between hormone treatments (85%) compared to controls (90%). There was also a treatment x time interaction (P < 0.05) for number of sperm lost in the backflow (2.1 ± 0.1 x 108), but by 8 h following AI, there was no effect on total sperm lost for the hormone treatments (38%) compared to C (54%). There was a trend (P = 0.10) for increased numbers of sperm in the uteri of hormone-treated gilts (6.0 ± 1.3 x 104) compared with C gilts (2.2 ± 1.3 x 104, but there was no effect of treatment on sperm numbers in the oviducts (3.2 ± 1.3 x 104). Within 0.5 h of AI, there was an increase in the frequency of contractions for PG compared with the other treatments (14.2 vs. 6.3/h, P < 0.005), however there was no effect on amplitude (54 mmHg) or duration (35 s) of contractions. The PR was not influenced by treatment and averaged 54% (P > 0.60), but total numbers of healthy fetuses were increased (P < 0.04) by PG (8.7) and tended (P = 0.06) to be increased for OT (8.4), but not for E (7.2) compared to C (5.8). Hormone addition to semen increased numbers of fetuses and this may be related to an alteration in the pattern of fluid and sperm loss after AI and a tendency for increased numbers of sperm in the anterior segment of the uterus. Therefore, in situations of lowered fertility, hormone addition could be a strategy to limit infertility in swine.
Key Words: Artificial Insemination • Contraction • Oxytocin • Pigs • Prostaglandins • Semen
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Introduction
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Addition of estrogens (Claus et al., 1989), PGF2 (Henze and Jurk, 1986; Pena et al., 2000), and oxytocin (Baker at al., 1968; Pena et al., 1998) to boar semen has been used to improve reproduction with AI. Estrogens are present in high concentrations in boar semen (Claus et al., 1990) and have been shown to stimulate myometrial contractions (Langendijk et al., 2002b) by causing the release of PGF2 from the endometrium (Claus et al., 1987). The addition of PGF2 to semen is reported to increase litter size, farrowing rate (Henze and Jurk, 1986; Pena et al., 2000), and myometrial contractions (Langendijk et al., 2002b), but the results have not always been consistent (Flowers and Esbenshade, 1993). Supplementation of oxytocin to semen has also increased farrowing rates in some cases, but this effect was also inconsistent (Flowers and Esbenshade, 1993). However, Pena et al. (1998) reported that litter size and farrowing rates were improved by oxytocin during the periods of seasonal infertility in the summer months. Collectively, the effect of the addition of these hormones to semen yielded inconsistent results, which has raised the question of the need, efficacy, and mechanism of action for these hormones for improving fertility with AI. Therefore, to address the potential benefit of hormone addition to extended semen for improving the efficiency of AI, this study was based on a sensitive fertility model using only a single low-dose insemination (0.5 x 109 sperm/80 mL) to mimic a situation of compromised fertility. The study determined the effect of one of three hormones supplemented in boar semen (estrogens, PGF2, or oxytocin) on: 1) semen backflow following AI, 2) the number of sperm in the oviduct and anterior segment of the uterus 8 h after AI, 3) frequency, amplitude, and duration of uterine contractions following AI, and 4) pregnancy rate and litter size at 25 to 30 d postmating.
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Materials and Methods
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Experimental Design
Experiment 1.
This experiment evaluated the effect of hormone supplementation to extended semen on backflow (the volume of semen and number of sperm expelled from the vulva following AI), pregnancy and the number of fetuses at d 25 to 30, placental weight, and fetal weight. Prepubertal gilts (n = 120) 170 ± 10 d of age, with an average BW of 113 ± 10 kg, and of mixed genetics (Landrace-Yorkshire, Landrace-Yorkshire-Duroc, and Duroc) were given an injection of PG600 (400 IU of PMSG and 200 IU of hCG; Intervet Inc., Millsboro, DE) to induce estrus. Twelve hours after injection, a mature boar was brought to the gilts for fence-line estrus detection, which consisted of nose-to-nose contact for 10 min and back pressure applied from a technician. Estrus detection occurred every 12 h until the onset of estrus, which was determined as the first standing response to a boar using the back-pressure test. An animal had to display two consecutive standing responses to be considered in estrus. After standing estrus was confirmed, the gilts were randomly allotted to treatment based on age, genetics, and BW, and then moved into gestation stalls.
A proportion of the gilts were used to quantitate backflow after expressing estrus and assignment to treatment (n = 48). Gilts were assigned to receive a single insemination of 0.5 x 109 sperm in 80 mL of extender (Androhep Plus, Minitube of America, Verona, WI) 24 h after the onset of estrus. Gilts received an AI with semen containing no hormone (C, control, n = 12), 11.5 µg of estrogens (E, 5 µg of estradiol-17ß, 4.5 µg of estrone sulphate, and 2 µg of estrone, n = 11), oxytocin (OT, 4 IU, n = 12), or PGF2 (PG, 5 mg of Lutylase; Pharmacia Co., Peapack, NJ; n = 13). The semen used for the study was a pool from three commercially owned boars of known fertility. One hour before insemination, all gilts were checked to ensure that all animals were still in standing estrus when insemination was performed at 24 h after onset of estrus. The hormones were added to the semen immediately prior to insemination. All animals were inseminated with a spirette catheter (Minitube of America, Verona, WI) with semen 
72 h old.
Backflow of semen for each treatment was collected continuously using a modified foam tip catheter (Minitube of America, Verona, WI), which was locked in the cervix. The catheter was modified to extend approximately 5.0 cm distal of the vulva and was attached to a 50-mL conical centrifuge tube. Backflow samples were collected at 0, 0.25, 0.5, 1, 2, 4, and 8 h relative to insemination. During insemination (0 h), a 200-mL cup was held beneath the vulva to collect effluent. Backflow samples were collected and stored at 5°C and evaluated for volume and sperm number. Following AI, gilts were moved to 1.8 x 3.5-m pens and housed in groups of four to six until slaughter (n = 120). Reproductive tracts were collected at slaughter and assessed for ovulation rate (number of corpora lutea, CL), pregnancy, number of fetuses, fetal weight, and placental weight at 25 to 30 d after AI. The total number of fetuses was counted in addition to the number of degenerative and healthy fetuses. Degenerative fetuses were distinguished from healthy fetuses based on appearance and smaller weight (>1 g lower than the average of all fetuses) and appeared to be in the process of being absorbed.
Experiment 2.
This experiment evaluated the effect of hormone addition to semen on the number of sperm retained in specific sections of the reproductive tract following AI. Prepubertal gilts (n = 7 per treatment) were induced into estrus, checked for estrus, and inseminated similar to procedures used for Exp. 1. Backflow was also collected for 8 h to account for number of sperm lost before the tracts were obtained. After the last backflow sample was collected, animals were anesthetized with an i.m. injection of 2.5 mg/kg of BW of (each) tiletamine and zolazepam, ketamine, and xylazine (Fort Dodge Animal Health, Fort Dodge, IA). This was followed immediately by stunning using a captive bolt, and killing by jugular exanguination. The reproductive tracts were removed within 10 to 15 min after sedative injection. The oviduct was flushed in addition to a 10-cm segment of the anterior uterus, just adjacent to the uterotubal junction. The uterine horns were first ligated with umbilical tape placed 10 cm from the utero-tubal junction on the uterine side and a second ligature placed on the infundibulum side of the ampullary region of the oviduct. Hemostats were placed near both of the tape locations and a blunt-end 18-gauge needle was inserted near the uterotubal junction to perform a retrograde flush of the oviduct. The oviduct was infused with 10 mL of 0.9% (wt/vol) NaCl, and gently massaged to free any sperm lodged in crypts. To collect effluent, the opposite end of the oviduct was placed in a 15-mL centrifuge tube, the hemostats were removed, and the volume was massaged from the oviduct. The same procedure was followed for flushing the uterus, except that a 14-gauge needle was used to infuse fluids, and a 50-mL centrifuge tube was used as the collection container. Subsequent to flushing, all collections were centrifuged at 1,200 x g for 3 min and the supernatant was removed, leaving 1 mL in the tubes with the cellular pellet. The centrifuged tubes were placed in a -20°C freezer overnight to lyse any red blood cells. The next day, the pellet was resuspended and the concentration of sperm evaluated using a hemacytometer.
Experiment 3.
This experiment evaluated the effect of hormone supplementation to extended semen on the frequency, duration, and amplitude of uterine contractions. Sows (n = 3 per treatment) were checked for estrus in crates every 12 h starting 24 h after weaning using a mature boar. Once estrus was detected, sows were assigned to treatment based on lactation length, parity, and genetics. Uterine contractions were measured using a MIKRO-TIP catheter pressure transducer (Millar Instruments Inc., Houston, TX). The transducer was pressure calibrated using a mercury manometer according to the recommendations of the manufacturer. Twenty-four hours after the onset of estrus, a modified intrauterine catheter (IMV International Corporation, Maple Grove, MN) was placed into the uterine body just anterior to the cervix. The tip of the inner catheter was bored out with a 0.8-cm drill bit to enable the transducer to be inserted through the catheter into uterus. The position of the tip of the transducer in the uterus was determined as follows: As the catheter traversed the cervical pads (approximately 5 pads), there was considerable resistance on the catheter at each of the pads. However, once the catheter bypassed the last pad and entered the uterus, there was little to no resistance, and it was quite easy to move the transducer back and forth. This was confirmed and practiced in the lab using sow reproductive tracts. Therefore, for proper positioning, the transducer cable was marked at a specific distance from the tip, which allowed consistent positioning over time within an animal. Insertion and positioning of the transducer for recording contractions typically required less than 3 min/female.
Once in position, the transducer was attached to a physiograph to record uterine contractions for 1 h before AI and for 2 h after AI. The physiograph was calibrated with the pressure transducer to record contractions between 10 to 60 mmHg. The criteria for defining a contraction event was based on the amplitude deviation from baseline and the duration of time that pressure was above baseline. From the physiograph recording, a contraction event was defined by one of the following five criteria: 1) 1 SD above baseline (20 mm Hg) lasting 50 s, 2) 2 SD above baseline (30 mmHg) that lasted 40 s, 3) 3 SD above baseline (40 mm Hg) that lasted 30 s, 4) 4 SD above baseline (50 mm Hg) that lasted 20 s, or 5) 5 SD above baseline (60 mm Hg) that lasted 10 s. Events that lasted less than 10 s were not counted as a contraction. Contractions were predominately measured while the animal was lying down; however, the animals were free to stand, sit, or lay in their stalls. Peaks originating from animal movement were marked during recording for exclusion from analysis.
Semen Processing.
Sperm-rich fractions from boar ejaculates were collected from a commercial boar stud and semen from the same three boars was used for all experiments. After collection, the fractions were pooled and extended at a ratio of one to one with extender to preserve the semen until further analysis. In the laboratory, sperm concentration was adjusted to 0.5 x 109 fertile sperm/80 mL, based on motility, morphology, and viability tests.
Preparation of Hormone Treatments.
All hormones were added in liquid form to the 80 mL of semen in extender in a 100-mL plastic bottle immediately before insemination. The estrogen solution was previously reported by Willmen et al. (1991). Each hormone was acquired from Sigma-Aldrich (St. Louis, MO). The concentration of estradiol-17ß, estrone sulphate, and estrone was 6.25, 5.63, and 2.50 x 10-2 µg/mL, respectively. The gilts assigned to receive PG were treated with 5 mg of PGF2 per insemination dose (1 mL of a 5-mg/mL solution of Lutylase; Cheng et al., 2000; Pena et al., 2000), and the animals inseminated with oxytocin received 4 IU/insemination dose (0.2 mL of a 20-IU/mL solution; Pena et al., 1998).
Statistical Analysis
In Exp. 1, backflow of semen (mL) and the number of sperm in the backflow samples were analyzed as a completely randomized design. A repeated-measures, mixed-effects model with treatment (four treatment levels) and animal (n = 48) as explanatory variables was fitted using PROC MIXED (SAS Inst., Inc., Cary, NC). Pregnancy at d 25 to 30 (n = 120; 0 for no, 1 for yes) was used to calculate pregnancy rate (number of gilts pregnant/number of gilts mated). Total number of fetuses (n = 67), number of healthy and degenerative fetuses, embryo survival (fetuses x 100/number of CL), and placental and fetal weight (kg) were also analyzed using a completely randomized design in PROC MIXED. Explanatory variables (where appropriate) included treatment, ovulation rate (covariate), semen age (1, 2, or 3 d old), fetal age (covariate, 25 to 30 d), and their interactions. In Exp. 2, the sperm numbers from both oviducts and uteri from each animal were pooled and analyzed as a repeated-measures, mixed-effects model that included ovulation rate and sperm number lost during insemination (0 h) as covariates. Sperm numbers flushed were transformed (log 2) in order to normalize the data for analysis. In Exp. 3, the response variables (frequency, duration, and amplitude of uterine contractions) were analyzed using a repeated-measures, mixed-effects model including treatment, animal, and parity (1, 3, 4, 5, and 7). Baseline measurements for frequency, duration, and amplitude were recorded before insemination and were used as covariates in the analyses. For all response variables, a normal distribution and unstructured variance-covariance matrix was assumed for the repeated measurements. The suitability of the assumptions was evaluated by inspection of residual plots. In analyses used to compare means in the study, a Scheffe or Bonferonni test was used to adjust for multiple comparisons among means. All interactions included in the model that were not significant (P > 0.10) were removed from the final model.
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Results
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Experiment 1
For backflow, there was a treatment by time interaction (P < 0.001). During insemination (0 h), gilts receiving PG treatment lost three times more (26.0 mL) semen compared to controls (8.5 mL) and almost twice (13.7 mL) the volume of gilts receiving estrogen or oxytocin (P < 0.0001, Table 1
). There was no difference among treatments in backflow from 0.25 to 1 h after insemination (P > 0.70), but the control gilts lost 11.5 mL more than the other treatments. Between 1 and 2 h following AI, the volume of semen backflow was also not different among treatments, even though the estrogen (16.6 mL)-treated gilts lost > 10 mL more at this time than either the oxytocin and PG treatments (5.6 and 1.9 mL, respectively), and 5 mL more than controls. By the end of the 8 h following AI, there was no difference in the total volume of backflow even though the OT treatment expelled 12 mL less than the other treatments.
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Table 1. Least squares means for the effect of hormone supplementation (estrogens, oxytocin, or PGF2) to semen on the volume (milliliters) of semen expelled from the uterus during insemination and at intervals following insemination in giltsa
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Similar to volume, there was a treatment x time interaction (P < 0.05) for the total number of sperm lost in the backflow. During insemination, 19% of the total sperm inseminated (97.0 x 106 sperm) was lost for the PG treatment compared to 8% (40.0 x 106 sperm) for the controls (Table 2). The estrogen and oxytocin treatments lost an average of 14% of the sperm (77.5 x 106 sperm), but were not different from the PG or the control groups. By 8 h, the control group expelled 54% of the total sperm inseminated (270 x 106 sperm) compared to 34% (170 x 106 sperm) for PG, 38% (190 x 106 sperm) for estrogen, and 42% (210 x 106 sperm) for the oxytocin treatments.
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Table 2. Least squares means for the effect of hormone supplementation (estrogens, oxytocin, or PGF2) of semen on the total number of sperm expelled from the uterus during insemination and at intervals after insemination in giltsa
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At d 25 to 30 following AI, pregnancy rates (59.0 ± 0.1%) were not different (P > 0.60, Table 3) among hormone treatment groups. However the total number of fetuses (calculated from the total number of gilts pregnant and including both healthy and degenerative fetuses) was influenced by treatment (P < 0.04). The total number of fetuses (adjusted for number of CL) was greater (P < 0.02) for PG (10.2) and tended (P < 0.06) to be greater for oxytocin (10.0), but not for estrogen (9.3) compared to controls (7.5). The number of healthy fetuses was also influenced by treatment (P < 0.04). The number of healthy fetuses was increased (P < 0.04) by PG (8.7) and tended (P = 0.06) to be greater for oxytocin (8.4), but not for estrogen (7.2) compared to controls (5.8). Embryo survival (%) was influenced by ovulation rate (P < 0.05) and was not influenced by treatment, even though means were numerically higher in the hormone treatments compared to controls (Table 3). Embryo survival was observed to decrease 1.4% for each increase in the number of CL. Overall, there was no difference in ovulation rate (15 ± 2.7 CL) or number of degenerative fetuses (1.7 ± 0.5) among treatments (P > 0.10). However, there was a difference in placental weight among the treatments (P < 0.01). Placenta for the PG group averaged 22.3 ± 1.0 g compared to 24.6 ± 1.5 g for the other treatments. There was no difference in fetal weights among treatments (P > 0.50) and each fetus averaged 1.6 ± 0.1 g.
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Table 3. Least squares means for the effect of hormone treatment (estrogen, oxytocin, or PGF2) on pregnancy rate (PR), fetuses, embryo survival, placental weight, and fetal weight in giltsa
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Experiment 2
There was no difference (P > 0.20) in the number of sperm that were flushed out between the left and right oviducts or the left and right 10-cm segments of the anterior uterine horns, and therefore data from both oviducts and both uterine horns were pooled (Table 4). The volume of semen leakage at 0 h during insemination tended (P < 0.07) to be slightly correlated to sperm numbers retained in the anterior segments of the uterus (r = -0.30), but not the oviducts (P > 0.50) There was a trend (P = 0.10) for the hormone treatments to have more sperm (6.0 x 104 sperm) in the anterior uterine segments compared to the controls (2.2 x 104 sperm). However, there was no difference among treatments (P > 0.80) for the total number of sperm flushed from the oviducts (3.2 ± 1.3 x 104 sperm, Table 4).
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Table 4. Least squares means for the effect of hormone supplementation (estrogens, oxytocin, or PGF2) to semen on the total number of sperm flushed from the reproductive tract 8 h after insemination in giltsa
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Experiment 3
A representative trace of the uterine contractions obtained from a single sow are shown in Figure 1. Before insemination, sows averaged 5.6 ± 0.6 uterine contractions/h, with an average amplitude of 50.4 ± 2.1 mmHg and with an average duration of 41.2 ± 6.2 s. The frequency of contractions for the controls appeared to increase from 5.2/h before AI to 11.0/h in the 2 h following AI, but this was not significant (P > 0.10). Following AI, there was a treatment x time effect (P < 0.001) for frequency of uterine contractions (Figure 2a). At 0.5 h after insemination, the PG group had more than twice the number of contractions (P < 0.005) compared to the other treatments (14.2 vs. 6.5/h). At 1 h, and continuing until the second hour following AI, there was no difference in the number of contractions among treatments (P > 0.40). There was no effect of treatment (P > 0.10) on the duration (35.1 ± 5.4 s, Figure 2c) or amplitude (54.2 ± 2.1 mmHg, Figure 2b) of uterine contractions. However, there was an effect of time (P < 0.003) for the duration of uterine contractions. Similar patterns were observed for the changes in the duration of uterine contractions over time for all of the treatments. The duration of contractions was greater before insemination (39.8 s), and then decreased at 0.5 h after insemination (28.6 s) before increasing at 1 h following AI (37.4 s).
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Figure 1. Representative recording of myometrial contractions using an intrauterine pressure transducer in a sow immediately following insemination at 24 h after the onset of estrus. The sow was housed in a gestation stall and was not anesthetized.
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Figure 2. Effect of hormone supplementation to semen (estrogens, oxytocin, or PGF2) on the frequency (2a), amplitude (2b), and duration (2c) of uterine contractions before and after AI in sows. A single insemination with 0.5 x 109 sperm/80 mL was performed 24 h (0 h) after the onset of estrus. Sows in each treatment received semen supplemented with no hormone/80 mL (CONTROL, n = 3), 11.5 µg of estrogens (E)/80 mL (n = 3), 4 IU of oxytocin (OT)/80 mL (n = 3), or 5 mg of PGF2 (PG)/80 mL (n = 3) immediately before AI. There was a treatment x time interaction for frequency of uterine contractions, but not for amplitude of contractions (P > 0.10). There was no effect of treatment on duration of contractions (P > 0.10), but there was an effect of time (P < 0.003).
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Discussion
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Hormone addition to semen or injection into females before or during insemination has been reported in some cases to increase fertility in swine in summer months and in lower parity sows. In the present study, to evaluate the effect of hormone addition to semen, we used a model to simulate a situation of lowered fertility. The low number of fetuses (<6) and pregnancy rates (62%) in the control treatment indicated that a situation of compromised fertility was achieved with a single insemination of 0.5 x 109 sperm in prepubertal gilts induced into estrus. These results indicate that hormone addition to the semen did not improve pregnancy rates but did increase the total number of fetuses when compared to no hormone addition. However, the mechanism for this effect is unclear. With the exception of contraction frequency immediately following insemination with PG, it was not possible to detect any clear effect of hormone addition on characteristics for frequency, duration, or amplitude of uterine contractions during a 2-h period following administration of hormone in the semen. Although total sperm retained in the anterior uterine segment and oviduct together were not influenced by hormone addition, there was a trend for the total number of sperm retained in the anterior segments of the uterus alone to be increased by 10% or more for all of the hormone treatments compared to the controls. However, the increased sperm numbers in the uterus did not translate into increased numbers of sperm in the oviduct 8 h after insemination. These data indicate that in situations of lowered fertility, the addition of prostaglandin or oxytocin could aid in establishing uterine sperm reservoirs, which can minimize the risk of lowered litter size.
These results show a difference in the total number of fetuses and number of healthy fetuses in response to hormone addition to semen. Collectively, overall reproductive performance (number of gilts x pregnancy rate x number of healthy fetuses) for total numbers of healthy pigs produced for each treatment was 174.9 pigs for PG, 133.5 pigs for oxytocin, 120.5 for estrogen, and 104.0 pigs for the control treatment. Therefore, hormone addition to semen appears to have advantages for overall pig production. Yet the mechanism for the increase in pigs produced is unclear. This response appears unrelated to ovulation rate since both PG and estrogen have higher ovulation rates whereas oxytocin and the control treatments have lower ovulation rates.
In the current study, hormone supplementation did not improve sperm numbers established in the reservoirs of the reproductive tract compared to the controls. However, a trend was observed for the hormone treated gilts to have more sperm in the anterior segments of the uterus (approximately 6.0 x 104 sperm) compared to the controls (2.2 x 104 sperm). Unlike the results of the present study, Langendijk et al. (2002a) infused Cloprostenol and observed lowered sperm numbers in the uterus and reduced fertilization rates. It is not clear why the results differ between the studies, but Lutalyse in the present study tended to increase sperm numbers in the uterus and increase numbers of fetuses. It should be noted that sperm retrieval from the genital tract is expected to be less than 100% (Langendijk et al., 2002a) because sperm may remain lodged in crypts. This, along with the efficiency of sperm transport, is most likely responsible for the high variation observed within animal, treatment, and between studies. In the present study, the higher numbers of sperm flushed from the uterus indicate that the hormone treatments may have increased sperm transport or may have rendered a more suitable environment to sustain the viability of the spermatozoa. Despite the numerical increase in sperm numbers in the anterior uterine segments, the numbers of sperm in the oviduct were not different for all treatments. It appears that the anterior uterine segment of the uterotubal junction may hold a greater significance as a primary sperm reservoir based on the number of sperm collected. This also leads us to believe that access into the ampullary region may be restricted to the greater numbers of spermatozoa occupying the uterus. Support for this comes from Mburu et al. (1996), who inseminated sows with an average of 11.4 billion sperm either naturally or artificially. The authors reported that sperm numbers in the oviduct ranged from 13,000 to 79,000, depending on the time of collection in relation to ovulation. A higher number of sperm was collected during the peri- and postovulatory periods than during the preovulatory period. Further, First et al. (1968) inseminated sows with an average of 40 billion sperm and removed the oviducts 8 h after insemination and observed ~32,000 sperm in the oviducts, similar to the results of the present study, despite large differences in the numbers of sperm inseminated (40 billion vs. 0.5 billion). In another study, Viring and Einarsson (1981) naturally mated sows 12 to 24 h after the onset of estrus and then flushed the oviduct, uterotubal junction, and the uterus. At 6 h after insemination, there were 1.3 x 104 sperm in the oviduct, 4.2 x 106 sperm at the uterotubal junction, and 3.7 x 108 sperm in the uterus. Collectively, these studies suggest that the number of sperm in the oviduct is not proportional to the number inseminated. Therefore, the results from each of these studies indicate that sperm in the reproductive tract can vary due to the time of collection in relation to ovulation, methods of sperm retrieval, and the specific areas of the tracts that are flushed.
In an analysis of the sperm collected in the backflow after insemination, it appeared that hormone supplementation to AI doses prebreeding did not alter the total volume of semen or number of sperm expelled from the reproductive tract, even though there was a treatment x time effect for volume and sperm lost over time following AI. However, it was apparent that the hormone-supplemented treatments lost less sperm (190 x 106 sperm) compared to the controls (270 x 106 sperm), but this was not significant. In our lab (Willenburg et al., 2003), an insemination of 3 x 109 sperm/80 mL resulted in backflow losses of 79% of the total volume and 57% of the total sperm within an 8-h period. Sterverink et al. (1998) inseminated multiparous sows with 1, 3, or 6 x 109 sperm/80 mL and collected backflow for 2.5 h. They observed losses equal to 70% of the total volume and 25% of the inseminated sperm. In the current study, the hormone treated animals lost an average of 85% of the total volume and 38% of the total sperm, whereas the controls lost 90% of the total volume and 54% of the total sperm. The increased volume of semen and number of sperm lost in the backflow could have resulted from the smaller size of the genital tract of the gilts used in the experiment when compared to sows. This interpretation is supported by Baker and Degen (1972), who reported that the size of the uterus and the number of sperm collected in the backflow were correlated. Therefore, although not significant, it appears that hormone supplementation to semen numerically reduced the number of sperm lost from the reproductive tract compared to the controls. During insemination, the PG treatment lost a larger volume and number of sperm than the other treatments resulting in loss of more than 40% of the inseminated volume and 19% of the total sperm. However, by 8 h after AI, there was no treatment effect on total sperm loss, despite the oxytocin treatment expelling 12 mL less than the other three treatments. The estrogen treatment lost a higher volume at 2 h compared to the previous collection times. This could be the result of a delayed response for the supplementation of estrogen. Therefore, the effects of estrogen would not be expected to be observed immediately following AI.
The influence of treatment was also assessed through uterine contractions. Contractions were measured for 2 h after insemination during the period when the majority of semen backflow was observed. The data revealed that of the hormone supplemented groups, only prostaglandin clearly influenced uterine activity in multiparous sows. Within a few minutes of insemination, the frequency of uterine contractions increased almost three-fold (from 5 to 6 contractions/h to 14 contractions/h), whereas the number of contractions for the other treatments (changing from 5.6 to 6.4 contractions/h) was not influenced. The fast but transient increase in frequency of uterine contractions could explain why the PG treatment expelled more than 77% of the volume lost within the first 0.5 h after insemination, whereas in the other treatments, the majority of the semen was lost during the second hour after AI. This was similar to the observations of Langendijk et al. (2002a, b), who reported that Cloprostenol increased contractions and also increased reflux during insemination. However, unlike the present study, these authors also observed that Cloprostenol increased the amplitude of contractions and that estrogen increased the frequency of uterine contractions. The reasons for the differences between the studies may be attributed to the smaller numbers of sows (n = 3/treatment) used in the present study. However, the reported means for frequency and amplitude of contractions were generally similar, indicating that the methods for determining these criteria were similar. Claus et al. (1989) infused 10 mL of saline with physiological amounts of estrogens (5 µg of estradiol + 2 µg of estrone + 4.5 µg of estrone sulphate) into the uterus and reported an increase in the frequency of uterine contractions (from 14/h during the first hour to 31/h by the second hour). In the current experiment, sows in the estrogen treatment averaged 12.5 contractions/h during the first 2 h following AI. Contractions were not measured past 2 h after insemination, and therefore, the full impact of the estrogen treatment may have not been observed. However, there was a trend for an increase in the number of sperm flushed from the uterus for the estrogen treatment, and from this it may be presumed that perhaps there was an increase in uterine activity after 2 h. Zerobin (1968) stated that natural mating and artificial insemination led to an increase in the frequency and amplitude of uterine contractions. This was thought to occur as a result of the large volume of semen deposited in the uterus in conjunction with cervical stimulation. In our study, following insemination, all treatments exhibited an increase in the frequency (from 5.7 to 8.3 contractions/h) and a slight increase in the amplitude (50.8 to 55.5 mmHg) of contractions, but these differences were not significant. The duration of contractions was higher before AI, and then decreased at 0.5 h after AI and increased again by 1 h after treatment. These observations may help explain the fluctuating phases of sperm transport that have been reported in the pig (rapid and sustained phases). Reports indicate that sperm are found in the isthmus of the oviduct within minutes of insemination (Hunter and Hall, 1974), which could result from an increase in the duration of uterine contractions. Hunter (1981) also stated that within 1 to 2 h after insemination a functional sperm reservoir is established in the oviducts. Based on the findings from each of these studies, uterine activity in the pig is influenced by hormones during the period of standing estrus; however, the interpretation of these contractions is highly variable due to level of hormone supplementation, contraction criteria, and method used for measurement. Natural mating and AI have been reported to increase the frequency and amplitude of uterine contractions (Zerobin, 1968). Claus et al. (1990) stated that endogenous oxytocin is released by the sow in response to boar stimulation and pheromones. Therefore, the mechanism of response to each of the semen-supplemented treatments would suggest an increase in uterine contractions, which would facilitate a greater number of sperm to arrive in the oviduct. However, our results suggest that sperm transport was not significantly altered or limiting to fertility. Despite a difference of more than 37,000 sperm among treatments for sperm in the uterine reservoir there was no difference in the number of sperm in the oviduct.
In conclusion, the study was designed to examine the effect of hormone addition to semen using a situation for compromised fertility. A single low-dose (0.5 x 109 sperm/80 mL) AI in gilts induced into estrus results in low fertility. Our data suggests that hormone supplementation would have the greatest opportunity for improving fertility when risk of fertility failure was high. Even though the mechanism of action is unclear and many effects nonsignificant, hormone supplementation altered the pattern for fluid volume and sperm loss following AI. This may have allowed a trend for higher numbers of sperm to situate themselves in the anterior segment of the uterine horn. These occurrences were associated with a higher total number of pigs following the addition of PGF2 and oxytocin to semen when compared to no hormone in low fertility situations. Additional research may be needed to find the level of hormone supplementation to semen to provide the optimal response. However, the data indicate that during times of low fertility (summer months or low parity animals), supplementation of semen with prostaglandin or oxytocin may be the most cost-effective method to reduce the risk of low fertility.
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Implications
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The effect of hormone addition to semen has been observed when fertility is low, such as during summer or in lower parity sows. In this study, a single low dose artificial insemination simulated a situation of reduced fertility. Hormone addition to semen altered the pattern of fluid and sperm loss following artificial insemination. This was associated with a trend for an increase in sperm retained within the anterior segment of the uterus and with an increased total number of pigs. Prostaglandin (1 mL of 5 mg/mL of Lutalyse) and oxytocin (4 IU, 0.2 mL of 20 IU/mL solution) may be the best choices for minimizing situations of compromised fertility. From these findings, hormone addition to semen may be useful in situations where the risk of compromised fertility is high. However, before hormones are added to semen, efforts should be directed toward accurate detection of estrus, optimizing semen quality, and proper artificial insemination timing and technique.
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Footnotes
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1 This research was supported in part by the Illinois Council on Food and Agricultural Research (C-FAR) and the Department of Animal Sciences, University of Illinois. The animal care and use committee of the University of Illinois approved the protocol 99386 for use of animals in this experiment. 
2 The authors thank the University of Illinois Moorman Swine Research Farm staff for their excellent technical support and care of animals during this study and Prairie State Semen Inc., Champaign, IL, for their assistance with this study.
Received for publication August 1, 2002.
Accepted for publication November 14, 2002.
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Literature Cited
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Abstract
Introduction
