Jc Dominguez

Thawing boar semen in the presence of seminal plasma: Effects on sperm quality and fertility.

Causes of poor fertility after insemination of frozen-thawed (FT) sperm include reduced sperm viability at thawing and a shorter longevity of surviving sperm in the female genital tract due to sub-lethal damage. The present studies examined the effect of incubating thawed boar sperm in seminal plasma (SP) on sperm membrane integrity (viability), and motility in vitro (experiment 1), and fertility in vivo (experiment 2). For experiment 1, FT sperm from five individual boars and a sperm pool from these boars were thawed and incubated for 4 h in media containing 0%, 10%, or 50% autologous seminal plasma (individual boars) or pooled seminal plasma (sperm pool). At approximately 10 min (0 h) and again at 1 h, 2 h, 3 h, and 4 h, sperm populations were examined for percentage sperm viability and percent sperm motility. Each variable progressively decreased during the incubation period. Incubation in 50% SP increased percentages of live sperm (P < 0.0001) and percent sperm motility (P < 0.01) at all time points compared to incubation in either 0% or 10% SP. For experiment 2, multiparous Large white x Landrace sows (n = 82) each received 900 IU eCG at weaning and 750 IU hCG 80 h later to control time of ovulation. Sows were assigned on the basis of parity to be inseminated with pooled semen with or without SP from the boars used in experiment 1. Sows received 3 x 10(9) live fresh-extended sperm (n = 30) or FT sperm thawed in 80 mL BTS extender (n = 26) or 3 x 10(9) live FT sperm thawed in 80 mL BTS containing 50% SP (FT-SP; n = 26). Sows were inseminated at 36 h, and 42 h after hCG injection. Compared to sows receiving fresh semen, the pregnancy rate of FT inseminated sows tended (P = 0.06) to be lower with the FT-SP group being intermediate. Farrowing rates were not different (83.3%, 69.2%, and 65.4% for fresh, FT, and FT-SP, respectively). Inseminations with FT sperm were associated with a reduction in litter size (P < 0.05), which was not evident in the FT-SP group. Taken together, these data confirm an adverse effect of inseminating FT sperm on sperm quality and sow fertility but suggest that thawing FT sperm in 50% SP may partially alleviate these adverse effects.

Treatment of swine summer infertility syndrome by means of oxytocin under field conditions.

Endogenous oxytocin is released by the sow at the time of mating in response to stimulation by the boar, which may explain, at least partially, the importance of the relationship between the boars courting activity and the subsequent reproductive performance of the sow. The aim of this study was to determine the effects on reproductive performance of supplementing AI doses with exogenous oxytocin during the low fertility season. At an intensive piggery in northwest Spain 3 experimental groups were randomly formed and observed throughout the year. Group 1 sows were inseminated with semen supplemented with 4 IU oxytocin. Group 2 sows received 4 IU oxytocin injected through the vulvar lips mucosa at the time of insemination. Group 3 sows were inseminated without oxytocin and served as the controls. During the low fertility season the results for each group were as follows: farrowing rate 77.02, 56.25 and 54.39%, and litter size 10.77 +/- 0.28, 10.45 +/- 0.31 and 8.53 +/- 0.34 respectively. It is concluded that the addition of oxytocin to seminal doses just before AI is an easily applicable, effective method for increasing fertility and litter size during the summer months.

Laparoscopic surgery in a clinical case of seminoma in a cryptorchid dog

in east Africa has been revealed after having remained undetected throughout the period of the JP15 campaign and eight years of PARC. The exact location of this focus is uncertain but surveillance is concentrating on north eastern Kenya and Somalia. It is unlikely that wildlife in Tsavo National Park has acted as a reservoir to maintain the virus in Kenya. Serosurveillance of wildlife populations in north eastern Kenya has not implicated these species in the maintenance of the virus in a silent form. Virus of type 2 lineage isolated from an infected kudu produced only mild disease in Kenyan cattle (Wamwayi and others 1996) while another virus from eland was found to be generally mild in British cattle, although one infected animal died from classical rinderpest (C. Dunn, personal communication). Since this virus is highly virulent in certain wildlife species, wildlife acts as sentinels for the disease. In contrast, the RGK-1 isolate is highly virulent for domestic cattle (Liess and Plowright 1964, Wohlsein and others 1995) and so the pathogenic phenotype of the virus cannot be inferred from its genetic lineage. The endemic focus of the type 2 virus is most likely to be in the border areas between north eastern Kenya and Somalia. In some of these areas rinderpest control is rudimentary or non-existent and it is possible that the virus has persisted unnoticed for over 30 years. Very few sequence changes are needed to alter the virulence of rinderpest virus; the genome of the Plowright vaccine strain differs by less than 0-55 per cent from the virulent virus from which it was derived (Baron and others 1996). Nothing is known concerning the molecular factors which determine the virulence/attenuation of different rinderpest virus strains and it is possible that changes in pathogenic phenotype can occur on passage through different animal species (Plowright 1963). Reversion to the mild form could be a means whereby the virus evades detection for many years. The ability to genetically manipulate morbilliviruses through rescue of live virus from DNA copies of their genomes (Baron and Barrett 1997) means that it will now be possible to address questions concerning virus attenuation and pathogenicity by directly altering virus genes. Only when the molecular basis of pathogenicity is understood will we be able to interpret the sequence data in a scientifically meaningful way. There is major concern over how such a notoriously severe dis-

Effect of vulvomucosal injection of PGF2α at insemination on subsequent fertility and litter size in pigs under field conditions

The aim of this study was to determine the effects of injecting 5 mg of PGF2alpha into the vulvar mucosa on the reproductive performance of sows maintained under field conditions. At an intensively managed piggery in northwest Spain, two experimental groups were formed randomly and observed throughout the year. The first group comprised sows receiving simultaneously, with every insemination, 5 mg of PGF2alpha injected into the vulvar lips. Group 2 sows received 1 ml of saline solution injected into the vulvar lips at insemination and served as the controls. The farrowing rates for each group were 78.46 and 54.39, while the litter sizes were 10.72 +/- 0.27 and 9.14 +/- 0.47 during the low fertility season (July-September). During the rest of the year (October-June), the farrowing rates were 83.91 and 80.93, while the litter sizes were 11.16 +/- 0.15 and 9.99 +/- 0.15. We conclude that injection of 5 mg of PGF2alpha into the vulvar lips at insemination is an effective method of compensating for the low fertility together with the decreased litter size of the summer months.

Effect of eCG or eCG Plus hCG on oestrus expression and ovulation in prepubertal gilts.

To meet weekly breeding targets, it is occasionally necessary to inject exogenous gonadotrophins to induce oestrus in prepubertal gilts. However, the gilt oestrus response to equine chorionic gonadotrophin (eCG) either alone or in combination with human chorionic gonadotrophin (hCG) can be unpredictable. The objective of the present study was to examine possible reasons for this unpredictability. Prepubertal gilts (90 kg and 153 days of age, n = 109) received an injection of either 600 IU eCG or a combination of 400 IU eCG and 200 IU hCG (PG600), or were non-injected controls, and were then exposed to a mature boar for 15 min daily for 7 days for oestrus detection. At the time of injection, real-time ultrasound revealed that the gilt ovaries had primarily 1-2 mm follicles. Blood samples were obtained at time of hormone injection (day 0) and at days 3, 7 and 10 for assay of serum progesterone concentrations. The oestrus responses by 7 days were 15.5%, 73.3% and 0%, for eCG, PG600, and control gilts, respectively (p < 0.001). The oestrus response improved (p < 0.05) with increasing body weight. Based on circulating progesterone levels, all oestrous gilts ovulated except for four of the PG600 gilts. Failure to express oestrus in PG600 gilts was not associated with a premature rise in progesterone.

Effect of gonadotropin treatment on estrus, ovulation, and litter size in weaned and anestrous sows

In the first of 2 experiments, we evaluated the effects on anestrous sows of pretreatment with FSH to stimulate the growth of small follicles, followed by eCG to stimulate the growth of medium follicles, estrus, and ovulation. In Exp. 2, we examined the effect of sows receiving 400 IU of eCG plus 200 IU of hCG (PG 600, Intervet/Schering Plough Animal Health, Boxmeer, the Netherlands) at weaning and then different doses and timing of supplemental hCG. In Exp. 1, a total of 87 multiparous Hypor sows deemed anestrus 7 d after weaning were assigned to intramuscular (i.m.) injection of 1) PG 600, 2) eCG (600 IU), 3) pretreatment with 87.5 IU of FSH on d 7 and 8 plus eCG on d 9, or were 4) noninjected controls. Sows had daily boar contact for 15 d after weaning for estrus detection. Blood samples were obtained on d 9 and 19 and assayed for progesterone to determine ovulation status. The weaning-to-estrus interval, number of sows in estrus and ovulating, farrowing rate, and litter size were not different (P > 0.1) in treated groups compared with controls. In Exp. 2, a total of 247 Hypor sows were assigned at weaning by parity (1 and 2 or > or = 3) to receive 1) an i.m. injection of PG 600, 2) PG 600 supplemented with 100 IU of hCG injected either concurrently or after 24 h, 3) 200 IU of hCG after 24 h, or 4) no injection (controls). Sows were exposed to boars daily for 7 d. After treatment of parity 1 and 2 sows, all gonadotropin-treated groups had an increased (P < 0.05) number of sows in estrus compared with the control group; weaning-to-estrus interval, farrowing rates, and litter size were unaffected (P > 0.1). After treatment of parity > or = 3 sows, there was no treatment effect on the estrous response and weaning-to-estrus interval; compared with control and PG 600-treated sows, farrowing rate was decreased (P < 0.05) for sows receiving 200 IU of hCG after 24 h. There was no effect (P > 0.1) of treatment on litter size. We conclude that gonadotropins can be used to increase estrus response in weaned sows, but that hCG treatment subsequent to PG 600 may be detrimental to sow fertility in parity > or = 3 sows.

Effect of prior FSH treatment on the estrus and ovulation responses to eCG in prepubertal gilts.

The objective of this study was to determine the effect of pre-treatment of prepubertal gilts with FSH on the estrus and ovulatory responses to eCG injection at two ages. A total of 149 prepubertal Hypor gilts were selected at 150 days (n=76) or 180 days (n=73) of age and assigned to injection of 400 IU eCG plus 200 IU hCG (PG600), 600IU eCG alone (Folligon), pre-treatment with 72 mg FSH (Folltropin) administered as 6 x 12 mg injections at 12 h intervals with 600 IU Folligon 12h after last FSH injection, or non-injected controls. To facilitate detection of estrus, gilts were exposed to a mature boar for 15 min daily for 7 days. To determine ovulatory responses, blood samples were obtained on the day of injection and 10 days later and assayed for progesterone content. Following treatment at 150 days, one control gilt (5.3%) was deemed estrus but ovulation did not occur. Compared to treatment with Folligon alone, PG600 injection tended (P=0.1) to increase the estrus response (52.6% compared with. 26.3%) and increased (P<0.01) the ovulatory response (89.5% compared with. 47.4%). The estrous response in gilts pretreated with Folltropin was intermediate (42.1%) but the ovulatory response (47.4%) was the same as for Folligon alone. Following treatment at 180 days, two control gilts (10.5%) were deemed estrus and ovulation did occur in these gilts. There was no difference between hormone-treated groups for estrus or ovulatory responses, although the ovulatory response of PG600-treated gilts tended (P=0.1) to be greater than for the Folligon-treated group (89.5% compared with 66.7%), with Folltropin-pretreated gilts being intermediate (76.5%). These data demonstrate that the estrus and ovulatory responses of gilts were greater for PG600 than for Folligon and that while responses to PG600 were not affected by gilt age, for the combined Folligon groups, estrous response (P<0.02) and ovulatory response (P<0.05) improved with increased gilt age.

Effect of Single Layer Centrifugation Porcicoll (70%, 80% and 90%) or supplementation with reduced glutathione, seminal plasma and bovine serum albumin on frozen-thawed boar sperm

Selecting the optimal sperm population is essential for success with reproductive techniques. Porcicoll (formerly Androcoll-P) is a colloid formulation for selection of high-quality boar spermatozoa by single layer centrifugation (SLC). To date, most studies have been carried out with fresh semen and large volumes. We carried out 2 experiments to test the use of Porcicoll for thawed boar semen in small volumes. In Experiment 1, cryopreserved semen doses were thawed, split in 200-μL aliquots and layered on 1mL of Porcicoll 70%, 80% or 90%, or buffer without colloid. We assessed sperm recovery (the proportion of the loading dose that appeared in the pellet, %), and the physiology of the selected spermatozoa (flow cytometry: Viability, apoptotic changes, capacitation, mitochondrial activity, intracellular reactive oxygen species). The most suitable proportion was Porcicoll 80%, allowing acceptable sperm recovery (16.9±4.2%, compared to 70% (35.4%±3.0, p<0.001) and 90% (8.2%±3.0, P=0.001), and improved quality (mitochondrial activity: Porcicoll 80%: 77.7±1% vs Control: 60.3±0.7%, P<0.05). In Experiment 2, we compared 3 supplements to Porcicoll 80%: 500mM reduced glutathione (GSH), 20% seminal plasma (SP) and 0.5% bovine serum albumin (BSA). Supplementation with GSH or BSA did not cause relevant changes relative to Control. In contrast, SP induced membrane and acrosomal changes resembling capacitation, which might preclude its use in some applications, and decreased recovery (5.5%±1.9 vs. 24.3%±1.2 Control; P<0.001). However, it could be useful prior to other applications such as in vitro fertilisation. Overall, Porcicoll is an effective colloid for isolating a high-quality population from thawed boar sperm, 80% being a balanced option for good recovery and high quality. Supplements could be useful depending on the proposed use of the spermatozoa.

Effect of hCG on Early Luteal Serum Progesterone Concentrations in PG600-Treated Gilts

Gilt oestrus and ovulation responses to injection of a combination of equine chorionic gonadotrophin (eCG) and human chorionic gonadotrophin (hCG) (PG600) can be unpredictable, possibly reflecting inadequate circulating LH activity. The objective of this study was to determine the effect of PG600 followed by supplemental hCG on gilt ovarian responses. In experiment 1, 212 Hypor gilts (160 day of age) housed on two farms in Spain received intramuscular (i.m.) injections of PG600 (n = 47), or PG600 with an additional 200 IU hCG injected either concurrently (hCG-0; n = 39), or at 24 h (hCG-24; n = 41) or 48 h (hCG-48; n = 45) after PG600. A further 40 gilts served as non-injected controls. Ovulation responses were determined on the basis of initial blood progesterone concentrations being <1 ng/ml and achieving >5 ng / ml 10 d after the PG600 injection. The incidence of ovulating gilts having progesterone concentrations >30 ng/ml were recorded. During the study period, 10% of control gilts ovulated whereas 85-100% of hormone-treated gilts ovulated. There were no significant differences among hormone groups for proportions of gilts ovulating. The proportions of gilts having circulating progesterone concentrations >30 ng/ml were increased (p < or = 0.02) in all hCG treated groups compared with the PG600 group. In experiment 2, a total of 76 Hypor gilts at either 150 or 200 days of age were injected with PG600 (n = 18), 400 IU eCG followed by 200 IU hCG 24 h later (n = 20), PG600 followed by 100 IU hCG 24 h later (n = 17), or 400 IU eCG followed by 300 IU hCG 24 h later (n = 21). Blood samples were obtained 10 days later for progesterone assay. There were no effects of treatment or age on incidence of ovulation, but fewer 150-day-old gilts treated with PG600 or 400 IU eCG followed by 200 IU hCG had progesterone concentrations >30 ng / ml. We conclude that hCG treatment subsequent to PG600 treatment will generate a higher circulating progesterone concentration, although the effect is not evident in older, presumably peripubertal, gilts. The mechanism involved and implications for fertility remain to be determined.

Surgical correction of a canine preputial deformity

or CPV-2b by both methods. All 22 isolates classified as CPV-2a by REA were also classified as CPV-2a by mAb. Of the 20 isolates typed as CPV-2b by REA, however, 18 typed as CPV-2a by mAb. Sixteen cPv isolates from Australia (collected from 1979 to 1993) were antigenically typed (Table 3). All three isolates from 1979 were typed as CPV-2. Of six isolates from 1980, two were typed as CPV-2 and four as CPV-2a. The seven isolates from 1990 onwards were classified as CPV-2a. These results provide confirmation that there has been a shift from the original CPV-2 virus to new variants. With the exception of one isolate, collected in 1990 from a closed beagle colony, cPv2 type virus was not detected in any of the isolates collected after 1984 from the USA, Germany or the UK by mAb or REA, demonstrating the replacement of the original CPV-2 type virus by CPV-2a and CPV-2b. By both methods of classification the majority of isolates from the USA were of the CPV-2b type and this is in agreement with the observations of Parrish and others (1991). The German isolates were approximately evenly distributed between CPV-2a and CPV-2b. A similar observation was made by De Ybanez and others (1995) using antigenic analysis of 13 Spanish isolates. In the case of the UK isolates, however, while by REA they were evenly distributed, by antigenic analysis the majority were found to be of the CPV-2a type. It is interesting to note from the antigenic analysis of 16 Australian isolates, that there was a similar shift from CPV-2 to CPV-2a between 1979 and 1993 (Table 3). Both mAb and REA can be used to classify canine parvoviruses although the typing of current field viruses by these two methods does not agree in all cases. From the immunological point of view the use of mAbs may have more significance as it detects changes in the important epitopes of the capsid, although REA can provide additional information for epidemiological studies. The two major field types CPV-2a and CPV-2b when classified by REA differed in that viruses typed as CPV-2b were 60 base pairs (bp) smaller than those typed as CPV-2a (Greenwood and others 1995). This difference is attributed to a deletion of one copy of a 60 bp tandem repeated sequence that spans the end of the capsid protein gene and the non-coding terminus. Such a deletion has been described by Parrish and others (1985, 1988) for a CPV-2a type isolate (CPV-31) that contained only one copy of the 60 bp repeat but did not show a change in antigenic type. This change observed by REA would not be expected to affect the antigenic sites of the virus as it falls outside the capsid protein gene where the antigenic determinants reside. In the light of this, it is interesting that there was agreement in the typing of the majority of isolates from Germany and the USA by both methods whereas the majority of UK isolates typed as cPv2b by REA were typed as CPV-2a by mAb. Of 46 UK isolates examined, only two were typed as CPV-2b by mAb, indicating that there is no rapid evolvement from CPV-2a to CPV-2b in the UK.