Effect of boar exposure at time of insemination on factors influencing fertility in gilts

The effect of boar exposure during artificial insemination (AI) on semen backflow, fertilization, and embryo quality was evaluated. Gilts (approximately 170 d) were induced into estrus with PG600, and ovulation was synchronized using hCG 72 h later. Estrus detection was initiated after PG600 and continued at 12-h intervals. At estrus, gilts were allotted to receive boar exposure (BE, n = 20) or no boar exposure (NBE, n = 20) during AI. Gilts receiving NBE were identified to be in estrus prior to AI and the boar was then removed for 1 h, whereas gilts in the BE group received 15 min of exposure during AI. Insemination occurred in crates at 12 and 24 h after onset of estrus with 3 x 10(9) sperm/80 mL. Backflow was collected continuously with samples taken at time 0, (during AI), and at 0.25, 0.5, 0.75, 1, 2, 4, and 8 h after first and second AI. The effect of treatment was evaluated for time of insemination (min), backflow (mL), and sperm in backflow samples. Oviducts were flushed 2 d after first AI to evaluate the effect oftreatment on fertilization rate, accessory sperm numbers on embryos (scored 1 to 5), and embryo quality. There was no effect of first or second AI; therefore, data were pooled. Average duration of AI was 3.7 +/- 0.2 min and was not influenced by BE (P < 0.10). However, during the initial stage of AI, BE reduced the volume of semen (18.6 vs 32.4 +/- 3 mL) and the number of sperm lost (0.8 vs 1.3 +/- 0.15 x 10(9) sperm) compared to NBE (P < 0.05). There was a treatment x time effect (P < 0.05) for volume of backflow. By 45 min, the BE gilts lost more volume (9.0 vs 3.6 mL) compared to the NBE group, but sperm loss did not differ. Between 1 and 8 h after AI, neither volume nor sperm loss was influenced by treatment. By 8 h, total leakage (65 vs 63 mL) and total sperm loss (1.6 x 10(9) vs 1.8 x 10(9) sperm) were not influenced by BE (P > 0.10). However, more accessory sperm (P < 0.01) were found on embryos for the NBE (> or = 11 sperm/embryo) compared to BE embryos (< or = 10 sperm/embryo). Despite this observation, percentages of fertilized embryos (99.5 +/- 0.5 %) and number of embryos (11.5 +/- 0.1) were not different (P > 0.10). In conclusion, AI in the presence of a mature boar did not affect total semen leakage, sperm loss, fertilized embryos, or embryo quality. The importance of boar exposure during insemination was evident from less leakage during insemination, but had no effect on fertility; this suggests that the elimination of boar exposure during AI may not be deleterious to reproductive performance.     

The effect of spermatozoa and seminal plasma on leukocyte migration into the uterus of gilts.

Yorkshire x Landrace gilts were used to determine the effect of spermatozoa and seminal plasma on postbreeding uterine leukocyte influx. Estrus detection was performed with a boar at 12-h intervals following synchronization with 400 IU eCG and 200 IU of hCG. All gilts were AI once, 24 h after the detection of estrus following random assignment to a 2x2x3 factorial arrangement of treatments (sperm or sperm-free AI doses), AI dose medium (seminal plasma or PBS), and lavage time following AI. Gilts were treated with sperm (5x10(9) spermatozoa; SPZ; n = 30) or sperm-free (SF; n = 30) doses containing either 100 mL of seminal plasma (SP; n = 15/treatment) or PBS (n = 15/treatment). Uterine lavage was performed once on each gilt (n = 20/time) at one of three times after AI (6, 12, or 36 h) to determine the total number of uterine leukocytes. The leukocytes consisted predominately (92 to 99%) of polymorphonuclear neutrophilic granulocytes (PMN). There was an AI x medium interaction on uterine PMN numbers. The number of uterine PMN recovered from gilts inseminated with sperm suspended in PBS was greater than the number of PMN recovered from the uterine lumen of gilts inseminated with sperm in SP, SP alone, or PBS alone (P<.05). Furthermore, SP accelerated the rate of uterine clearance when suspended with sperm cells during the first 36 h following AI (P<.05). These results indicate that seminal plasma suppresses PMN migration into the uterus following breeding and enhances the rate of disappearance of uterine inflammation.     

Uterine immune reaction and reproductive performance of sows inseminated with extended semen and infused with pooled whole dead semen

The objective of this study was to investigate the effect of infusing whole dead semen (WDS) after AI with diluted commercial semen on uterine inflammatory reaction and embryonic survival rate in gilts. Sixty Yorkshire-Landrace gilts were assigned at their second estrus to one of the following AI treatments: 1) commercial semen adjusted to 1 x 10(9) sperm cells (S1) per dose, followed by an infusion of 80 mL of WDS (S1-WDS); 2) S1 followed by an infusion of 80 mL of Beltsville Thawing Solution (S1-BTS); 3) commercial semen adjusted to 3 x 10(9) sperm cells (S3) per dose, followed by an infusion of 80 mL of BTS (S3-BTS); and 4) a negative control group, in which gilts received two infusions of 80 mL of BTS (BTS). Two days after the first AI, eight gilts from Groups 1, 2, and 4 were slaughtered and reproductive tracts were collected. One horn was cut open longitudinally along the antimesometrial aspect and endometrial samples were taken and immediately frozen for analysis of messenger RNA (mRNA) abundance for inflammatory cytokines and growth factors. The other horn was flushed with 20 mL of PBS, and the contents of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1) were determined by ELISA. On d 25 after AI, gilts from Groups 1, 2, and 3 were slaughtered and their reproductive tracts were collected to evaluate the number of fetuses and corpora lutea. On d 2 after the first AI, only TGF-beta1 was detected in the flush of all gilts, and no difference was observed between S1-WDS, S1-BTS, and BTS gilts. Endometrial levels of IFN-gamma and interleukin (IL)-6 mRNA were marked in all gilts, but they were not affected by the AI treatments, whereas the mRNA abundances for IL-1 and IL-2 were negligible. Infusions of WDS or BTS after a fertile AI did not affect IGF-I, IGF-I receptor, or IGF-II mRNA levels compared with gilts infused with BTS only, whereas the mRNA abundance for the IGF-II receptor was decreased (P < 0.05) in WDS-infused gilts. In gilts inseminated with S1 doses, infusion of WDS did not affect the number of live embryos. Although infusions of WDS did not affect the mRNA level and secretion of the cytokines measured and did not improve embryonic survival rates, further studies are needed to better understand the influence of semen composition on the uterine response after mating.     

Fertilization and blastocyst development in oocytes obtained from prepubertal and adult pigs

Polyspermic fertilization and embryo quality are important issues for the in vitro production of pig embryos. We hypothesized that oocyte donor (prepubertal gilt vs. sow) affects polyspermy and blastocyst development in vitro and that the sexual maturity of the oocyte donor affects the response to sperm concentration in the fertilization medium. In Exp. 1, oocytes of sows and gilts were mounted and stained 12 h after insemination to provide fertilization data. In Exp. 2, putative embryos were developed in vitro to 144 h post-insemination before mounting. In both experiments, cumulus-oocyte complexes (COC) were collected from ovaries of prepubertal gilts and adult sows. Sperm were added after maturation of COC for 40 to 44 h. Sperm from two boars at 0.5 to 5.0 x 10(6) sperm/mL was used for insemination. More (P < 0.01) monospermic fertilizations were observed in oocytes derived from gilts than for oocytes from sows. There were fewer (P < 0.02) penetrated sperm per fertilized oocyte in oocytes from gilts compared with sows. There were effects of semen donor (boar) on the percentage of monospermic (P < 0.01) and polyspermic (P < 0.002) fertilizations, and on the number of penetrated sperm/fertilized oocyte (P < 0.02). In Exp. 2, cleavage and blastocyst formation was evaluated at 2 and 6 d postinsemination, respectively. More (P < 0.001) blastocysts developed from sow-derived oocytes than from gilt-derived oocytes. More (P < 0.05) total cells per blastocyst were observed in embryos from sow-derived oocytes than from gilt-derived oocytes. Semen donor affected the percentage of oocytes cleaving (P < 0.02), and a boar x sperm concentration interaction affected (P < 0.05) the incidence of blastocyt formation. Results indicate that sexual maturity of the donor is not responsible for the high incidence of polyspermy in porcine in vitro fertilization. However, blastocyst development is improved by the use of oocytes from sows rather than from prepubertal gilts.     

Enhancement of ovulation rate in gilts by increasing dietary energy and administering insulin during follicular growth

Two experiments were conducted to examine influences of dietary energy and insulin on ovulation rate and patterns of luteinizing hormone (LH), follicle stimulating hormone (FSH), glucose, insulin and estradiol in gilts during 6 d before estrus. In Exp. 1, 36 gilts were given altrenogest for 14 d to synchronize estrus. In a factorial arrangement, gilts were fed one of two levels of dietary energy (5,771 or 9,960 kcal metabolizable energy (ME)/d), and given one of two levels of porcine insulin (0 or .1 IU/kg body weight iv every 6 h). Dietary treatments began 4 d before and insulin treatments began 1 d after the last day of altrenogest, respectively, and lasted until 24 h after estrus. Main effect means for number of corpora lutea were 14.0 +/- 1.3 and 17.6 +/- .9 for 5,771 and 9,960 kcal ME (P less than .05), and 14.6 +/- 1.0 and 17.0 +/- .9 for 0 and .1 IU insulin (P less than .05). Number of LH peaks on d 3 was greater for gilts that received 9,960 kcal than 5,771 kcal (3.3 +/- .2 vs 2.7 +/- .2; P less than .05), and for .1 than 0 IU insulin (3.2 +/- .2 vs 2.7 +/- .2; P less than .05). During the first 24 h of sampling, concentrations of LH and FSH were greater (P less than .05) in gilts receiving 9,960 kcal ME plus insulin than for other treatment combinations. Concentrations of estradiol were not affected by treatments. In Exp. 2, two formulations of insulin were evaluated for influence on ovulation rate. All gilts received altrenogest and 9,960 kcal ME/d as in Exp. 1. Then on the first day after altrenogest, seven gilts each received short-acting insulin (as in Exp. 1), long-acting insulin (zinc suspension, 1.0 IU/kg body weight every 18 to 24 h), or served as controls. Ovulation rates were increased (P less than .05) by both insulin preparations (15.6, control; 19.1, short-acting; 18.5, long-acting; SE = 1.2). Concentrations of LH tended to be greater after short-acting insulin, but differences were not significant (P = .13). We conclude that increases in ovulation rate produced by dietary energy and insulin are not necessarily accompanied by changes in gonadotropins or estradiol.     

Accessory sperm: their importance to fertility and embryo quality, and attempts to alter their numbers in artificially inseminated cattle.

Accessory sperm number and its relationship to fertilization and embryo quality was evaluated in cattle after nonsurgical recovery of ova or embryos 6 d after insemination. Efforts to alter accessory sperm number per ovum included 1) blockage of retrograde sperm loss at insemination using a modified insemination device, 2) elevated sperm number per inseminate (40 x 10(6) vs 20 x 10(6], and 3) alteration in semen quality (percentage of viable and morphologically normal sperm in the inseminate). None of these efforts affected accessory sperm number per ovum or embryo. However, blockage of retrograde semen flow for 3 h or use of semen of below-average quality (decreased percentage of viable and morphologically normal sperm) resulted in significant decreases in number of viable embryos and increases in number of degenerate embryos and unfertilized ova compared with conventional insemination (P less than .03) and use of semen with an average percentage of viable and morphologically normal sperm (P less than .06). Number of accessory sperm per embryo or ovum was positively related to fertilization and embryo quality (P less than .05). Mean accessory sperm +/- SD and the median value (in parentheses) for unfertilized ova, degenerate embryos, and embryos classified fair to poor and excellent to good were, respectively, .3 +/- .8 (0), 5.4 +/- 8.9 (1.0), 15.8 +/- 28.6 (3.5), and 16.9 +/- 29.5 (5.0). We conclude that efforts to improve accessory sperm numbers per embryo or ovum failed and that high variation and skewness of accessory sperm toward 0 may make median values more meaningful than means.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution and retention of spermatozoa with acrosomal and nuclear abnormalities in the cow genital tract

Two experiments were conducted to evaluate the fate of sperm following uterine insemination. In Exp. I, five pairs of Holstein cows were inseminated with egg yolk-Tris extended semen (approximately 1.0 X 10(9) sperm; .5 ml) from five ejaculates from a single bull that had high levels (approximately 70%) of morphologically abnormal sperm. Cows were slaughtered 12 h after insemination. The genital tracts were removed and promptly clamped into defined regions. Sperm were recovered by flushing with 2.9% sodium citrate buffer. Proportions of abnormal sperm in the various regions were compared with those in the inseminate. Sperm numbers were also determined from each region. Regions of the tract varied in number of sperm (P less than .001), proportions of knobbed acrosomes (P less than .001), tapered heads (P less than .001), protoplasmic droplets (P less than .001), tail abnormalities (P less than .029) and total abnormalities (P less than .002). A total of 63.5 +/- 6.4 X 10(6) sperm was recovered. These sperm were distributed throughout the tract as follows: vagina, 91.8%; cervix, 5.4%; uterine horns, 2.7%, and uterotubal junctions-isthmi, .04%. No sperm were recovered from ampullae. Because retrograde movement of sperm from the uterus occurred in Exp. I, we conducted Exp. II to determine the extent of sperm loss from the genital tract following insemination. Three pairs of Holstein cows were inseminated with .42 X 10(9) sperm (.5 ml; egg yolk-Tris extender) from the same bull used in Exp. I (three ejaculates). All discharged mucus and urine was collected for 12 h after insemination for recovery of sperm. Aspirates (approximately 1 ml) of mucus from the vagina were evaluated during the 12-h post-insemination period for numbers of sperm and leucocytes. Sperm were also recovered from the tract following slaughter (approximately 12 h) to determine retention. Overall, 73 +/- 3.7% of inseminated sperm were recovered. Components were: inseminate lost from the genital tract in discharged mucus, 60 +/- 4.6%; lost in urine, .06 +/- .02%; aspirated from the vagina, 4.4 +/- 1%; adhered to equipment, 1.3 +/- .3%, and retained in the genital tract, 6.5 +/- 1.6%. Predicted numbers of sperm contained in discharged mucus 2 h post-insemination were greater (P less than .009) than at subsequent hours.(ABSTRACT TRUNCATED AT 400 WORDS)     

Artificial insemination outcomes in beef females using bovine sperm with a detectable fertility-associated antigen

In this study, semen samples from 25 bulls that had passed a breeding soundness evaluation were analyzed for the presence or absence of a 31-kDa protein, known as fertility-associated antigen (FAA), on spermatozoal membranes. Eighteen bulls had FAA on sperm (FAA-positive) and seven were devoid of FAA on sperm (FAA-negative). A single ejaculate from each bull was extended and frozen with 25 to 30 x 10(6) sperm in .5-mL straws. Crossbred replacement heifers (n = 865) were estrus-synchronized and artificially inseminated either at timed AI or 12 h after they were detected in estrus. Mature cows (n = 285) were inseminated 12 h after they were detected in estrus during a 45-d AI period. Pregnancy rates (pooled) to first AI service for females (n = 764) inseminated with FAA-positive sperm were 65.6% and were 49.7% for females (n = 386) inseminated with FAA-negative sperm (P < .005). Among the estrus-synchronized replacement heifers, pregnancy rates to synchronized AI service for heifers (n = 550) inseminated with FAA-positive sperm were 62% and were 45.7% for heifers (n = 315) inseminated with FAA-negative sperm (P < .005). These data indicate that pregnancy rates to first AI service at spontaneous and synchronized estrus are higher when using semen from bulls with detectable FAA on spermatozoal membranes compared to semen from bulls devoid of FAA on membranes. Fertility-associated antigen is an important determinant for fertility potential of sperm from bulls to be used in AI breeding programs.     

Assessing in vivo Fertilizing Capacity of Liquid-Preserved Boar Semen According to the 'Hanover Gilt Model'

The goal of this study was to determine the ability of the Hanover gilt model to assess in vivo fertilizing capacity of preserved sperm and to consider whether any modifications to this model were needed. This model evaluates the fertilizing capacity of semen based on the fertilization rate, the rate of normal embryos and the accessory sperm count of 3–5‐day embryos. Its distinguishing characteristics are the use of one‐time insemination of sperm in reduced numbers, of spontaneously ovulating gilts and of ovulation detection through ultrasound examination of ovaries. Reduced sperm numbers allow for an accurate evaluation of the fertilizing potential of different semen treatments, thereby avoiding the compensatory effect of doses calibrated to maximize fertility. The model's usefulness was assessed in a trial run designed to compare the fertilizing capacity of liquid boar semen diluted into two different extenders. The diluent, the boar and the backflow, had no significant effect on any of the parameters studied. Gilts inseminated less than 24 h before ovulation had a significantly higher (p < 0.01) fertilization rate and accessory sperm cell count (p < 0.05) than those inseminated more than 24 h before ovulation. Very good/good embryos from homogeneous litters (only very good/good embryos were present) had a significantly higher (p < 0.01) accessory sperm count than those from heterogeneous litters (at least one embryo was of a different quality and/or oocytes were present). Both very good/good and degenerated/retarded embryos from heterogeneous litters had low accessory sperm numbers. This suggests that accessory sperm count is significantly related to the quality of the litter, but not to the quality of the embryo within gilts. It can be concluded that the Hanover gilt model is sensitive enough to show fertility differences (in this study, those associated with in vivo ageing of semen), while using relatively few gilts and little time.     

Effect of age and physical or fence-line boar exposure on estrus and ovulation response in prepubertal gilts administered PG600

Boar exposure has been used for estrus induction of prepubertal gilts, but has limited effect on estrus synchronization within 7 d of introduction. In contrast, PG600 (400 IU of PMSG and 200 IU of hCG; Intervet, Millsboro, DE) is effective for induction of synchronized estrus, but the response is often variable. It is unknown whether boar exposure before PG600 administration might improve the efficiency of estrus induction of prepubertal gilts. In Exp. 1, physical or fence-line boar contact for 19 d was evaluated for inducing puberty in gilts before administration of i.m. PG600. Exp. 2 investigated whether 4-d boar exposure and gilt age influenced response to PG600. In Exp. 1, 150-d-old prepubertal gilts were randomly allotted to receive fence-line (n = 27, FBE) or physical (n = 29, PBE) boar exposure. Gilts were provided exposure to a mature boar for 30 min daily. All gilts received PG600 at 169 d of age. Estrous detection continued for 20 d after injection. In Exp. 2, prepubertal gilts were allotted by age group (160 or 180 d) to receive no boar exposure (NBE) or 4 d of fence-line boar exposure (BE) for 30 min daily before receiving PG600 either i.m. or s.c. Following PG600 administration, detection for estrus occurred twice-daily using fence-line boar exposure for 7 d. Results of Exp. 1 indicated no differences between FBE and PBE on estrus (77%), age at puberty (170 d), interval from PG600 to estrus (4 d), gilts ovulating (67%), or ovulation rate (12 corpora lutea, CL). Results from Exp. 2 indicated no effect of age group on estrus (55%) and days from PG600 to estrus (4 d). A greater (P < 0.05) proportion of BE gilts expressed estrus (65 vs. 47%), had a shorter (P < 0.05) interval from PG600 to estrus (3.6 vs. 4.3 d), and had decreased (P < 0.05) age at estrus (174 vs. 189 d) compared with NBE. Ovulation rate was greater (P < 0.05) in the BE group for the 180-d-old gilts (12.7 vs. 11.9 CL) compared with the NBE group. However, age group had no effect on ovulation (77%) or ovulation rate (12 CL). Collectively, these results indicate that physical boar contact may not be necessary when used in conjunction with PG600 to induce early puberty. The administration of PG600 to 180-d-old gilts in conjunction with 4 d prior fence-line boar exposure may improve induction of estrus, ovulation, and decrease age at puberty.     

Effect of Sperm Cryopreservation and Supplementing Semen Doses with Seminal Plasma on the Establishment of a Sperm Reservoir in Gilts

Frozen-thawed (FT) boar sperm have a reduced fertile life, due in part to a capacitation-like status induced by cooling. Reversal of this cryocapacitation in vitro by exposure to boar seminal plasma (SP) has been demonstrated. The objective of these studies was to determine the effect of SP on the ability of FT sperm to create an oviductal sperm reservoir following artificial insemination (AI). In Experiment one, 35 pre-pubertal gilts were injected (IM) with 400 IU eCG plus 200 IU hCG to induce oestrus. At detection of oestrus, gilts were inseminated with 3 x 10(9) live sperm, either fresh (FS; n = 13), FT (n = 10), or FT supplemented with 10% v/v SP (n = 12). Gilts were killed 8 h later, their reproductive tracts recovered and the uterotubal junctions (UTJs) flushed to recover sperm. Fewer (p < 0.01) sperm were recovered following FT, compared to FS, inseminations, and there was no evident effect of SP. In Experiment two, 30 pre-pubertal gilts received IM injections of 1000 IU eCG followed by 5 mg pLH 80 h later to control time of ovulation. Gilts were inseminated with 3 x 10(9) live FS sperm (n = 6), FT sperm (n = 15) or FT sperm plus 10% SP (n = 9) at 12 h before ovulation and then sacrificed 8 h later. The UTJs were dissected and flushed for sperm recovery. Fewest (p < 0.001) sperm were recovered following FT insemination and there was no evident effect of SP. These data demonstrate that the size of the sperm reservoir is markedly reduced in gilts inseminated with FT sperm. However, the lack of effect of SP suggested that either it did not reverse cryocapacitation or that such a reversal does not impact the in vivo ability to create a sperm reservoir.     

Effects of P.G. 600 on the onset of estrus and ovulation rate in gilts treated with Regu-mate.

Three experiments assessed the onset of estrus and ovulation rate in gilts treated with gonadotropins after the withdrawal of an orally active progestin. In Exp. 1, all cycling gilts received the progestin (Regu-mate; Intervet America Inc., Millsboro, DE) at a rate of 15 mg/d for 18 d. Twenty-four hours after the last feeding of Regu-mate, 32 gilts received an i.m. injection of 400 I.U. PMSG and 200 I.U. hCG (P.G. 600, Intervet America, Inc.), and 32 gilts received an i.m. injection of deionized water. The percentage of gilts displaying estrus < or = 7 d (P = 0.64) and the injection-to-estrus interval (P = 0.37) were similar for P.G. 600-treated gilts (93.8% and 4.1 +/- 0.1 d) and controls (90.6% and 4.3 +/- 0.1 d). Ovulation rate was greater (P < 0.01) in P.G. 600-treated gilts (28.8 +/- 1.1) compared with controls (17.4 +/- 1.1). In Exp. 2, 58 cycling gilts received Regu-mate (15 mg/d) for 18 d. Twenty-four hours after Regu-mate withdrawal, gilts received i.m. P.G. 600 or water (n = 29/treatment). Gilts were bred via AI 12 and 24 h after first detection of estrus. The percentage of gilts displaying estrus < or = 7 d (P = 0.45) and the injection-to-estrus interval (P = 0.27) were similar for P.G. 600-treated gilts (82.7% and 4.0 +/- 0.1 d) and controls (89.7% and 4.2 +/- 0.1 d). Ovulation rate was greater (P < 0.01) in P.G. 600-treated gilts (26.2 +/- 1.8) compared with controls (18.1 +/- 1.7). Pregnancy rate (P = 0.71) and the number of live embryos at d 30 postmating (P = 0.40) were similar for P.G. 600-treated gilts (91.7% and 15.6 +/- 1.2) and controls (88.5% and 14.1 +/- 1.2). In Exp. 3, prepubertal gilts (142.6 +/- 0.7 d of age) received Regumate (15 mg/d) (n = 20) or a control diet not including Regu-mate (n = 20) for 18 d. Twenty-four hours after Regu-mate withdrawal, all gilts received i.m. P.G. 600. The percentage of gilts displaying estrus < or = 7 d (P = 0.49) and the P.G. 600-to-estrus interval (P = 0.69) were similar for Regu-mate-fed gilts (95% and 4.3 +/- 0.2 d) and controls (88.9% and 4.2 +/- 0.2 d). Ovulation rate was similar (P = 0.38) for Regu-mate fed gilts (16.6 +/-1.6) and controls (14.4 +/- 1.8). In cycling gilts, administration of P.G. 600 after withdrawal of Regu-mate increased ovulation rate, but not litter size at d 30 postmating. There was no beneficial effect of Regu-mate pretreatment on the response to P.G. 600 in prepubertal gilts.     

Factors influencing boar sperm cryosurvival

Optimal sperm cryopreservation is a prerequisite for the sustainable commercial application of frozen-thawed boar semen for AI. Three experiments were performed to identify factors influencing variability of postthaw sperm survival among 464 boar ejaculates. Sperm-rich ejaculate fractions were cryopre-served using a standard freezing-thawing procedure for 0.5-mL plastic straws and computer-controlled freezing equipment. Postthaw sperm motility (assessed with a computer-assisted semen analysis system) and viability (simultaneously probed by flow cytometry analysis after triple-fluorescent stain), evaluated 30 and 150 min postthaw, were used to estimate the success of cryopreservation. In the first experiment, 168 unselected ejaculates (1 ejaculate/boar), from boars of 6 breeds with a wide age range (8 to 48 mo), were cryopreserved over a 12-mo period to evaluate the predictive value of boar (breed and age), semen collection, transport variables (season of ejaculate collection, interval between collections, and ejaculate temperature exposure), initial semen traits, and sperm quality before freezing on sperm survival after freezing-thawing. In Exp. 2, 4 ejaculates from each of 29 boars, preselected according to their initial semen traits and sperm quality before freezing, were collected and frozen over a 6-mo period to evaluate the influence of interboar and intraboar ejaculate variability in the survival of sperm after cryopreservation. In Exp. 3, 12 ejaculates preselected as for Exp. 2, from each of 15 boars with known good sperm cryosurvival, were collected and frozen over a 12-mo period to estimate the sustainability of sperm cryosurvival between ejaculates over time. Boar and semen collection and transport variables were not predictive of sperm cryosurvival among ejaculates. Initial semen traits and sperm quality variables observed before freezing explained 23.2 and 10.9%, respectively, of the variation in postthaw sperm motility and viability. However, more that 70% of total variance observed in postthaw sperm quality variables among ejaculates was explained by boar. This indicates that boar is the most important (P < 0.001) factor explaining the variability among ejaculates in sperm cryosurvival, with most (14 of the 15 boars in Exp. 3) showing consistent (P > 0.05) sperm cryosurvival over time.     

Omega-3 fatty acids in the gravid pig uterus as affected by maternal supplementation with omega-3 fatty acids

Two experiments evaluated the ability of maternal fatty acid supplementation to alter conceptus and endometrial fatty acid composition. In Exp. 1, treatments were 1) the control, a corn-soybean meal diet; 2) flax, the control diet plus ground flax (3.75% of diet); and 3) protected fatty acids (PFA), the control plus a protected fish oil source rich in n-3 PUFA (Gromega, JBS United Inc., Sheridan, IN; 1.5% of diet). Supplements replaced equal parts of corn and soybean meal. When gilts reached 170 d of age, PG600 (PMSG and hCG, Intervet USA, Millsboro, DE) was injected to induce puberty, and dietary treatments (n = 8/treatment) were initiated. When detected in estrus, gilts were artificially inseminated. On d 40 to 43 of gestation, 7 gilts in the control treatment, 8 gilts in the PFA treatment, and 5 gilts in the flax treatment were pregnant and were slaughtered. Compared with the control treatment, the flax treatment tended to increase eicosapentaenoic acid (EPA: C20:5n-3) in fetuses (0.14 vs. 0.25 +/- 0.03 mg/g of dry tissue; P = 0.055), whereas gilts receiving PFA had more (P < 0.05) docosahexaenoic acid (DHA: C22:6n-3) in their fetuses (5.23 vs. 4.04 +/- 0.078 mg/g) compared with gilts fed the control diet. Both the flax and PFA diets increased (P < 0.05) DHA (0.60, 0.82, and 0.85 +/- 0.078 mg/g for the control, flax, and PFA diet, respectively) in the chorioallantois. In the endometrium, EPA and docosapentaenoic acid (C22:5n-3) were increased by the flax diet (P < 0.001; P < 0.05), whereas gilts receiving PFA had increased DHA (P < 0.001). The flax diet selectively increased EPA, and the PFA diet selectively increased DHA in the fetus and endometrium. In Exp. 2, gilts were fed diets containing PFA (1.5%) or a control diet beginning at approximately 170 of age (n = 13/treatment). A blood sample was collected after 30 d of treatment, and gilts were artificially inseminated when they were approximately 205 d old. Conceptus and endometrial samples were collected on d 11 to 19 of pregnancy. Plasma samples indicated that PFA increased (P < 0.005) circulating concentrations of EPA and DHA. Endometrial EPA was increased (P < 0.001) for gilts fed the PFA diet. In extraembryonic tissues, PFA more than doubled (P < 0.001) the EPA (0.13 vs. 0.32 +/- 0.013 mg/g) and DHA (0.39 vs. 0.85 +/- 0.05 mg/g). In embryonic tissue on d 19, DHA was increased (P < 0.05) by PFA (0.20 vs. 0.30 +/- 0.023 mg/g). Supplementing n-3 PUFA, beginning 30 d before breeding, affected endometrial, conceptus, and fetal fatty acid composition in early pregnancy. Dynamic day effects in fatty acid composition indicate this may be a critical period for maternal fatty acid resources to affect conceptus development and survival.     

Influence of Sperm Cell Dose and Post-insemination Backflow on Reproductive Performance of Intrauterine Inseminated Sows

This study was carried out to evaluate the effects of the sperm cell dose and semen backflow on the pregnancy rate and number of embryos of sows inseminated once at 0-24 h before ovulation, using an intrauterine technique. The results were analysed from a total of 211 sows assigned to three groups inseminated with doses of 0.25 x 10(9) (T1), 0.5 x 10(9) (T2) and 1.0 x 10(9) (T3) spermatozoa. Semen backflow was observed in 95% of the females (143/151) evaluated for this purpose. The percentage of semen backflow is close to two-third of the volume and the percentage of sperm is around 15% of the infused sperm dose. Intrauterine insemination can be successfully performed provided that at least 0.5 billion of sperm cell dose is infused at an interval of 0-24 h before ovulation.     

Optimizing the use of sex-sorted sperm in timed artificial insemination programs for suckled beef cows

Three experiments were designed to evaluate methods to optimize the use of sex-sorted sperm in timed AI (TAI) programs for suckled beef cows. In all 3 experiments, suckled Bos indicus cows were synchronized using an intravaginal progesterone (P4) device during 8 d and a 2.0-mg injection of intramuscular estradiol benzoate (EB) at device insertion. The females received PG and eCG (300 IU) at P4 device removal and 1.0 mg of EB 24 h later. The cows were inseminated 60 to 64 h after P4 device withdrawal. All cows had their ovaries scanned by transrectal ultrasound at TAI to indentify and to measure the largest follicle (LF) present. In Exp. 1, a total of 853 cows had their LF classified as <9 mm or ≥9 mm at the time of TAI; these cows were then randomly assigned to 4 groups according to their LF diameter (<9 mm or ≥9 mm) and the type of sperm used (sex-sorted or non-sex-sorted). There was an interaction (P = 0.02) between the type of sperm and LF diameter beginning at TAI[non-sex-sorted ≥9 mm = 58.9%a (126/214); non-sex-sorted <9 mm = 49.5%b (106/214);sex-sorted ≥9 mm = 56.8%ab (134/236); and sex-sorted <9 mm = 31.2%c (59/189), a≠b≠c = P < 0.05]. In Exp. 2, suckled cows (n = 491) were classified immediately before TAI as having displayed estrus or not (estrus or no estrus) between P4 device removal and TAI. These cows were randomly assigned to 4 groups according to the occurrence of estrus and the type of sperm (sex-sorted or non-sex-sorted). There were effects of the occurrence of estrus (P = 0.0003) and the type of sperm (P = 0.05) on pregnancy per AI [P/AI; no estrus, non-sex-sorted = 43.6% (27/62); estrus, non-sex-sorted = 58.5%; (107/183); no estrus, sex-sorted = 33.9% (21/62), and estrus, sex-sorted = 50.0% (92/184)]; however, no interaction between the occurrence of estrus and type of sperm was observed (P = 0.87). In Exp. 3, a total of 200 suckled cows presenting LF ≥9 mm at TAI were randomly assigned to receive sex-sorted sperm deposited into the uterine body (n = 100) or into the uterine horn ipsilateral to the recorded LF (n = 100). No effect of deeper AI on P/AI was found (P = 0.57). Therefore, the LF diameter at TAI and the occurrence of estrus can be used as selection criteria to identify cows with greater odds of pregnancy to receive sex-sorted sperm in TAI programs. In addition, performing TAI with sex-sorted sperm deeper into the uterus did not alter the pregnancy results.     

Deep-freezing of boar semen in plastic film 'cochettes'

The motility and membrane integrity of spermatozoa from nine boars frozen with a programmable freezing machine in plastic bags, 'cochettes', and in 'maxi-straws', in total doses of 5 x 10(9) spermatozoa/5 ml with glycerol (3%) used as cryoprotectant, were assessed after thawing. A computer-based cell motion analyser was used to evaluate sperm motility, while the integrity of the plasmalemma was assessed with fluorescent supravital dyes (C-FDA/PI). The fertilizing capacity of the semen frozen in the two containers was investigated by inseminating (AI) gilts. Pregnancy was monitored by Doppler-ultrasound, and the numbers of corpora lutea and viable embryos counted at slaughter, between days 30 and 38 after AI. The cochettes sustained the overall procedure of freezing/thawing (FT), with 30 min post-thaw (PT) sperm motility being significantly higher than for straws, 46.9 vs. 39.5%. The only significant difference in motility patterns detected when comparing the packages was a higher sperm velocity (VCL) in cochettes at 30 min PT. However, percentages of FT-spermatozoa with intact membranes, detected with the supravital probes, were higher in maxi-straws than in cochettes, 46.8 vs. 43.0% (P < 0.05). There were no significant differences found in fertilizing capacity between spermatozoa frozen in maxi-straws and those frozen in cochettes. The results indicate that although the deep-freezing of AI-doses of boar semen in large plastic bags is feasible, problems such as their inconvenient size for storage and inconsistent thawing must be solved before this type of container can be used for the commercial cryopreservation of boar semen.     

Luteinizing hormone release after administration of the gonadotropin-releasing hormone agonist Fertilan (goserelin) for synchronization of ovulation in pigs

The generic GnRH agonist, Fertilan (goserelin), was tested for the ability to induce an LH surge and ovulation in estrus-synchronized gilts. Three experiments were performed to 1) examine the effect of various doses of Fertilan on secretion of LH in barrows, to select doses to investigate in gilts (Exp. 1); 2) determine doses of Fertilan that would induce a preovulatory-like rise of LH in gilts (Exp. 2); and 3) determine the time of ovulation after Fertilan treatment (Exp. 3). In Exp. 1, 10 barrows were injected on d 1, 4, 7, 10, and 13 with 10, 20, or 40 microg of Fertilan; 50 microg of Gonavet (depherelin; GnRH control) or saline (negative control); and sequential blood samples were collected for 480 min. There was a dose-dependent stimulation (P < 0.05) of LH release. Maximal plasma concentrations of LH (LH(MAX)) were 2.1 +/- 0.2, 4.1 +/- 0.3, 2.6 +/- 0.4, and 3.4 +/- 0.3 ng/mL after 10, 20, and 40 microg of Fertilan and 50 microg of Gonavet, respectively, and duration of release was 78 +/- 9, 177 +/- 12, 138 +/- 7, and 180 +/- 11 min, respectively. Fertilan doses of 10 and 20 microg were deemed to be the most suitable for testing in gilts. In Exp. 2, 12 gilts received (after estrus synchronization with Regumate and eCG) injections of 10 or 20 microg of Fertilan or 50 microg of Gonavet 80 h after eCG to stimulate a preovulatory-like LH surge and ovulation. An LH surge was induced in 3 of the 4 gilts in both of the Fertilan groups and in all of the Gonavet-treated gilts. Characteristics of induced release of LH did not differ among groups: LH(MAX), 5.0 +/- 0.9 vs. 4.6 +/- 1.8 vs. 6.6 +/- 1.1 ng/mL; duration, 11.7 +/- 2.0 vs. 12.3 +/- 2.2 vs. 14.3 +/- 0.5 h; interval from GnRH injection to LH(MAX), 4.0 +/- 2.0 vs. 6.7 +/- 1.3 vs. 5.8 +/- 1.6 h. In Exp. 3, estrus-synchronized gilts were injected with 20 microg of Fertilan (n = 8) or 50 microg of Gonavet (n = 4), and the time of ovulation was determined by repeated endoscopic examination. Time of ovulation ranged from 34 to 42 h postGnRH; however, ovulation occurred earlier in the Gonavet compared with the other groups (P < 0.05). Results of these experiments indicate that 1) barrows are an appropriate model for determining GnRH doses that can be effective in inducing a preovulatory-like LH surge in females; 2) the generic GnRH agonist Fertilan, at doses of 10 to 20 microg, can stimulate an LH surge in gilts, with subsequent ovulation; and 3) Fertilan at doses of 10 and 20 microg should be examined further for use in fixed-time insemination protocols.     

Within-herd Use of Boar Semen at 5°C, with a Note on Electronic Monitoring of Oestrus

A system was designed to allow a small swine farm in a northern latitude to use its own boars for artificial insemination (AI) conveniently. Semen was collected twice weekly for 3 day use (days 0, 1 and 2), extended in an egg yolk extender and stored at 5°C. Farm personnel were trained to manage the entire AI programme. For simplicity all semen collected was used for insemination. In the first test 47 gilts and 15 sows were inseminated with semen from four boars. One boar was subfertile with a farrowing rate of 36%. The averages for the other boars ranged from 71 to 100%. Then semen was collected from seven boars and all was used to inseminate 70 gilts and 55 sows with 3 × 10⁹ or more sperm. Overall 63% farrowed an average of 10.1 piglets per litter. Litter size for sows was 1.5 piglets larger than for gilts. There was no difference in farrowing rate when more than 3 × 10⁹ sperm were inseminated. The feasibility of initiating a complete AI programme within a small herd using herd boars was established. However, selection of the boars, use of only high quality semen, and experience with detecting oestrus was required to increase the farrowing rate. The use of various agents to protect sperm against cold shock below 15°C is worthy of further investigation. A new type of electronic probe, which measures the conductivity of cervical mucus, could be helpful if a boar is not available for conventional detection of oestrus.     

Technical note: Artificial vagina vs a vaginal collection vial for collecting semen from rams.

The time required to train rams to an artificial vagina (AV) makes collecting semen from large numbers of rams difficult. To manage this problem, we developed a glass, round-bottomed, 1.9-cm i.d. x 9.8-cm long vaginal collection vial (VCV). Three experiments were conducted to determine whether the VCV affected 1) semen volume per collection, 2) percentage of motile spermatozoa, 3) forward progressive motility score before and after extension and after freezing and thawing, and 4) our ability to collect semen from untrained rams. A soft rubber cap with a hole in the center was used to cover the VCV. A VCV was inserted into the vagina of an estrual ewe, and a monofilament line attached to the VCV was clipped to the wool near the vulva. Rams were joined with unrestrained ewes in a pen until they ejaculated into the VCV. In Exp. 1, five rams trained to an AV were used in a switchback design with four collection periods. During each period (1 d), semen was collected with an AV and a VCV. Immediately after collection, semen volume and sperm motility were quantified. Semen was extended with an aloe vera gel-based diluent at a 1:4 dilution rate, motility was quantified again, and semen was frozen. At 1 h after freezing, semen was thawed and sperm motility was quantified. Ejaculate volume (mean = 0.7 mL) and all measures of motility after collection were similar (P > 0.05) for the two collection methods. In Exp. 2, 10 rams trained to an AV were used in a switchback design with five collection periods (period = 3 d). On d 1 and 3 of each period, an AV and a VCV were used to collect semen. Collection method did not affect (P > 0.05) ejaculate volume (mean = 1.0 mL), percentage of motile cells, or forward progressive motility score. In Exp. 3, 51 untrained rams were used in a switchback design with a single collection period (2 d). Semen was collected with an AV and a VCV. Ability to collect an ejaculate and time required for collection were recorded. The likelihood of collecting semen from untrained rams was greater (P < 0.01) using a VCV (mean = 31.4%) than using an AV (mean = 9.8%). Collection method did not affect (P > 0.05) ejaculate volume (mean = 0.8 mL), percentage of motile cells, or forward progressive motility score. We concluded that a VCV could be used to collect semen from rams that are not trained for semen collection without decreasing ejaculate volume or sperm motility.