Field fertility of frozen boar semen: a retrospective report comprising over 2600 AI services spanning a four year period.

Worldwide, greater than 90% of sows are inseminated with fresh semen. Less than 1% is inseminated using frozen semen. Albeit, frozen semen is an effective technology for the transfer of genes between breeding pyramids and also to reliably provide semen for planned matings. Little information exists on the long term use of frozen boar semen in commercial pork production operations. The objective in the present study was to assess application of frozen semen throughout a 4 year period comprising more than 2600 AI services. The frozen semen sourced from a boar stud in Manitoba, Canada. All artificial insemination (AI) occurred on a single 1800 sow farm in Indiana, USA. The sperm-rich fraction was collected and only those collections having ≥80% motility and ≤15% abnormalities were further processed. Semen was prepared for cryopreservation using Androhep(®) CryoGuard™, packaged in 0.5mL French straws (average 500 million total sperm per straw) and frozen using a programmable freezer (IceCube™). For each frozen ejaculate, a post-thaw quality check was performed. Ninety eight percent of the ejaculates that were frozen showed at least a 50% post-thaw motility and were approved for shipment. For AI, eight straws were thawed (to achieve at least 2.0×10(9)motile sperm) and diluted with 60mL of extender pre-warmed to 26°C. Within 2-5min of thawing, the sows or gilts were inseminated via intra-cervical deposition using a standard AI pipette. Sows and gilts were inseminated three times PM/AM/PM and AM/PM/AM, respectively. Of 2696 recorded services, 2122 (78.7%) of the females farrowed. The mean (±SD) total number piglets born were 12.5 (±3.9). A progressive improvement of fertility over time was observed mainly due to adaptive procedures associated with an introduced technology. In summary, acceptable fertility is possible with frozen semen and has merit for application as a reproductive management tool.

Computer-assisted semen analysis and its utility for profiling boar semen samples.

Achieving and maintaining a successful swine AI program depends on a number of factors, including accurate semen evaluation, typically sperm motility, morphology and concentration. Computer-Assisted Semen Analysis or CASA (i.e., image analysis with a phase-contrast microscope and computer measurements of motion parameters) objectively evaluates sperm motion characteristics, morphology and concentration. A total of 3077 semen collections were evaluated with CASA (on the day of collection), and a semen dose subset was used for single-sire AI of 6266 females over 6 months. Fertility data from these inseminations were fitted with models including farm/stud, line, boar, parity, mating week, semen age at mating and boar age at mating. The residuals from these models showed no correlation for any CASA semen unique motion parameter, which could be due to the level of sperm concentration, the number of inseminations per estrus, and the low number of females mated per boar. Future studies to expand CASA/fertility analysis need to address these constraints and may include analysis of extended boar semen after storage for 1 week.

Effect of epidermal growth factor and insulin-like growth factor I on porcine preantral follicular growth, antrum formation, and stimulation of granulosal cell proliferation and suppression of apoptosis in vitro.

The object of this study was to investigate the role of epidermal growth factor (EGF) and IGF-I in the regulation of preantral follicular growth, antrum formation, and granulosal cell proliferation/ apoptosis. Porcine preantral follicles were manually dissected and cultured for up to 8 d in Waymouth's (Exp. 1) or alpha-minimum Eagle's essential medium (Exp. 2 and 3) supplemented with 10 microg/mL of transferrin, 100 microg/mL of L-ascorbic acid, and 2 mU/mL of ovine FSH, in the presence (Exp. 1 and 3) or absence (Exp. 2) of 7.5% fetal calf serum. According to the experimental protocol, IGF-I (0, 1, 10, or 100 ng/mL; Exp. 1), or IGF-I (50 ng/mL), EGF (10 ng/mL) and EGF+IGF-I (Exp. 2 and 3) were added to the culture media. In Exp. 1, follicles exhibited a concentration-dependent response (P < 0.05) to IGF-I, with the highest rates of granulosal cell proliferation, follicular integrity, and recovery rate of cumulus cell-oocyte complexes and lowest incidence of apoptosis occurring at the highest IGF-I dose. In Exp. 2 serum-free medium, granulosal cell proliferation was low (1 to 5%), irrespective of whether EGF and/or IGF-I were present and cellular apoptosis was increased (P < 0.05) on d 4 and 8 in the EGF+IGF-I group compared with the addition of either factor alone. In Exp. 3, granulosal cell proliferation was high in all follicles cultured in serum-containing medium for the first 3 d, but fell sharply (P < 0.05) on d 4, except in media containing IGF-I. Collectively, EGF and IGF-I increased granulosal cell proliferation, decreased apoptosis, and promoted follicular antrum formation. These results may provide useful information for developing a preantral follicular culture system in which the oocytes are capable of fertilization and embryonic development.

Oviduct-specific glycoprotein modulates sperm-zona binding and improves efficiency of porcine fertilization in vitro.

Oviduct-specific glycoprotein (OGP) displays estrus-associated regional and temporal differences in expression and localizes to the zona pellucida, perivitelline space, and plasma membrane of oviductal oocytes and embryos, suggesting that it may have a role in regulation of fertilization and/or early embryonic development. The aims of this study were to evaluate the effect of exogenous OGP on in vitro fertilization (IVF) and embryo development in the pig using a defined serum-free culture system. In vitro-matured porcine oocytes were incubated with homologous OGP (0, 1, 10, 20, and 40 microg/ml) for 3 h and then washed prior to IVF. Exposure of oocytes to 10 or 20 microg/ml porcine OGP (pOGP) significantly reduced the incidence of polyspermy compared with the control (P < 0.01) while maintaining high penetration rates. When oocytes, spermatozoa, or both were preincubated with 10 microg/ml pOGP prior to IVF, the incidence of polyspermy was similarly reduced (P < 0.01) by all three treatments without affecting penetration rates. The ability of spermatozoa to undergo calcium ionophore-induced acrosome reaction was similar with or without exposure to pOGP. However, significantly fewer spermatozoa (P < 0.01) bound to the zona pellucida when oocytes were preincubated with pOGP. To evaluate the effect of pOGP on embryo development, embryos were cultured in pOGP-supplemented medium for 48 h or 144 h. Both transient and continuous exposure to pOGP significantly enhanced cleavage and blastocyst formation rate compared with the control (P < 0.01). These data demonstrate that exposure of either in vitro-matured oocytes or spermatozoa to pOGP decreased polyspermy and spermatozoa binding while maintaining high penetration rates of pig oocytes fertilized in vitro. Furthermore, pOGP exerted an embryotrophic effect independent of effects demonstrated on spermatozoa and oocytes at fertilization.

Use of in vitro fertilization and embryo transfer to circumvent infertility caused by an inherited imperforate vagina in mice.

An autosomal recessive mutation (ipv) causing infertility in homozygous females (ipv/ipv) because of imperforate vaginae was discovered in a line of mice selected for low lean tissue mass as a proportion of body weight. The aim of this study was to determine if the mutation could be propagated in offspring following embryo transfer of oocytes collected from mutant females and fertilized in vitro with sperm from males known to carry the gene (ipv/?). Caudal epididymal sperm were incubated with cumulus-enclosed oocytes for 8-10 hr in tissue culture medium 199 + 5% fetal calf serum + 0.4% bovine serum albumin. Oocytes possessing at least two pronuclei were transferred to recipient CD-1 females which had been mated 24 hr earlier to vasectomized males. A total of 683 oocytes was collected from 27 superovulated mutant females. A large proportion of the oocytes was abnormal as evidenced by cytoplasmic fragmentation (259/683, 38%). Seventy-eight percent (331/424) of the normal oocytes were fertilized and 181 of these were transferred to 10 recipients. Six of 10 recipients delivered 38 offspring (24 females, 14 males). Fifty-eight percent (14/24) of the female offspring displayed an imperforate vagina. The results demonstrate that in vitro fertilization and embryo transfer can be used for propagating a mutant gene that causes infertility in females.

Observations on the cooling and cryopreservation of pig oocytes at the germinal vesicle stage.

This study examined the viability of pig oocytes at the germinal vesicle stage following cooling or cryopreservation. Cumulus-intact oocytes (n = 641) were collected from slaughterhouse pig ovaries and used in two experiments. In Exp. I the viability of 1) control, 2) cryoprotectant control (CC, 1.5 M glycerol/.5 M sucrose), 3) cooled (0 degrees C) and 4) cryopreserved (-196 degrees C) oocytes was assessed after no incubation or a 24-h incubation. Survivability was judged by morphological appearance, trypan blue exclusion and fluorescein diacetate staining. Survival rate of control oocytes (90%; based primarily on morphological appearance of the cumulus) incubated 0 h was greater (P less than .05) than that of all other groups, whereas survival rate of -196 degrees C oocytes (57%) was less (P less than .05) than that of all other groups. However, vital staining of 0 degrees C and -196 degrees C oocytes showed 0% survival rate as evidenced by trypan blue uptake and lack of fluorescence. The cumulus cells surrounding oocytes that were stored at 0 degrees C or -196 degrees C survived freezing as evidenced by trypan blue exclusion and intense fluorescence. Similar differences among treatment groups were found for oocytes incubated 24 h. Exp. 2 examined the temperature at which oocytes became sensitive to cooling. Oocyte death occurred when oocytes were cooled to 15 degrees C or lower. These results demonstrate that pig oocytes at the germinal vesicle stage did not survive cooling to 15 degrees C or below. When assessing the viability of cryopreserved cumulus enclosed oocytes it is important to use vital stains in conjunction with morphological appearance.

Influence of calcium on the true acrosome reaction and capacity of bull spermatozoa to penetrate oocytes in vitro.

A series of experiments were conducted to determine the role of Ca in several physiological functions of bovine spermatozoa. For spermatozoa incubated in the absence of Ca for up to 24 h, motility was not different from those incubated with Ca. For spermatozoa incubated in the continuous presence of Ca, true acrosome reaction values were 0% at 0 h, 1.5% at 6 h, and 6.0% at 12 h. Spermatozoa incubated in vitro for up to 12 h in the absence of Ca did not undergo a true acrosome reaction; however, when Ca was added during incubation, a synchronous true acrosome reaction was induced within 10 min (0% at 0 h, 8.5% at 6 h, and 8.5% at 12 h). When spermatozoa were preincubated in the presence or absence of Ca for 6 h, then added to zona-intact dead bovine oocytes and incubation continued with and without Ca for 18 h, the number of spermatozoa binding to and penetrating each oocyte was greater when Ca was present. Also, the percentage of oocytes being penetrated was greater when Ca was present. These results indicate that: 1) Ca is not necessary for maintenance of spermatozoan motility; 2) Ca is required for the induction of a true acrosome reaction among a population of spermatozoa; 3) Ca is able to induce the synchronous true acrosome reaction in a low percentage of spermatozoa; and 4) Ca is important in spermatozoan binding and initiation of penetration of oocytes.

Staining procedure to detect viability and the true acrosome reaction in spermatozoa of various species.

A simple dual stain procedure (DS) for simultaneously determining sperm viability and acrosomal status is described. The DS includes the use of vital stain trypan blue to detect live and dead spermatozoa and Giemsa to detect the presence or absence of an acrosome. For staining, spermatozoa are washed, incubated with trypan blue, washed, dried onto slides, and subjected to Giemsa. Dead spermatozoa stain blue in the postacrosomal region while live spermatozoa remain unstained. The acrosome stains light purple-dark pink while acrosome-free sperm remain unstained. This staining pattern enables differentiation of spermatozoa which have undergone a true acrosome reaction (TAR) from those which have undergone a false acrosome reaction (FAR). Incubation of bull, boar, ram, and stallion spermatozoa for 60 minutes at 37 degrees C in the presence of calcium ionophore A23187 increased the proportion of spermatozoa undergoing a TAR in all species except the stallion. Incubation of bull spermatozoa for up to 24 hours at 37 degrees C resulted in a decrease over time in the percentage of live acrosome-intact spermatozoa and a simultaneous increase in the percentage of spermatozoa categorized as having undergone a TAR and FAR. The DS could be a useful technique in evaluating sperm viability and acrosomal status in fertilization and clinical studies.

In vivo capacitation and acrosome reaction of bovine sperm in estrous and diestrous cows.

A staining procedure which enables distinction between spermatozoa possessing a true and false acrosome reaction (AR) was utilized to assess the incidence of capacitation and the true AR of bull spermatozoa recovered from the uterine horns of estrous and diestrous cows. Results show that at 3 and 6 h post-insemination, approximately 14.5 and 31.5%, respectively, of the live spermatozoa recovered had undergone a true AR in the uterus of estrous cows. An increasing percentage of those spermatozoa recovered from estrous cows with time were categorized as undergoing a false AR. This suggests that spermatozoa underwent capacitation, a true AR, then died prior to fixation and staining, therefore being grouped as false acrosome-reacted. Few spermatozoa were observed to have undergone a true AR in diestrous cows. It is apparent from this study that individual spermatozoa undergo capacitation and a true AR at different times during incubation in utero in estrous cows.