Machine Learning Prediction of Adenovirus D8 Conjunctivitis Complications from Viral Whole-Genome Sequence.

Objective: To obtain complete DNA sequences of adenoviral (AdV) D8 genome from patients with conjunctivitis and determine the relation of sequence variation to clinical outcomes. Design: This study is a post hoc analysis of banked conjunctival swab samples from the BAYnovation Study, a previously conducted, randomized controlled clinical trial for AdV conjunctivitis. Participants: Ninety-six patients with AdV D8-positive conjunctivitis who received placebo treatment in the BAYnovation Study were included in the study. Methods: DNA from conjunctival swabs was purified and subjected to whole-genome viral DNA sequencing. Adenovirus D8 variants were identified and correlated with clinical outcomes, including 2 machine learning methods. Main Outcome Measures: Viral DNA sequence and development of subepithelial infiltrates (SEIs) were the main outcome measures. Results: From initial sequencing of 80 AdV D8-positive samples, full adenoviral genome reconstructions were obtained for 71. A total of 630 single-nucleotide variants were identified, including 156 missense mutations. Sequence clustering revealed 3 previously unappreciated viral clades within the AdV D8 type. The likelihood of SEI development differed significantly between clades, ranging from 83% for Clade 1 to 46% for Clade 3. Genome-wide analysis of viral single-nucleotide polymorphisms failed to identify single-gene determinants of outcome. Two machine learning models were independently trained to predict clinical outcome using polymorphic sequences. Both machine learning models correctly predicted development of SEI outcomes in a newly sequenced validation set of 16 cases (P = 1.5 × 10-5). Prediction was dependent on ensemble groups of polymorphisms across multiple genes. Conclusions: Adenovirus D8 has ≥ 3 prevalent molecular substrains, which differ in propensity to result in SEIs. Development of SEIs can be accurately predicted from knowledge of full viral sequence. These results suggest that development of SEIs in AdV D8 conjunctivitis is largely attributable to pathologic viral sequence variants within the D8 type and establishes machine learning paradigms as a powerful technique for understanding viral pathogenicity.

Passage of spermatozoa through the epididymis of the boar (Sus scrofa domesticus).

Previous studies of epididymal transit in the pig have had insufficient animal numbers to provide a comprehensive picture of a continuous process. The present study attempts to addresses this issue. Radioactively labeled thymidine was infused into the testicular arteries of 48 sexually rested young adult boars of the Goettingen Miniature Pig breed. Hemicastrations were performed in random order in 2 animals daily on Days 21-24 following thymidine injection and in 4 animals daily on Days 25-41 and 43, 45, 47 and 49. Sperm obtained from 12 epididymal sites between the proximal caput and distal cauda were autoradiographically examined to record the percentage of labeled sperm and labeling intensity at different times after thymidine infusion. An initial surge of labeled spermatozoa emerged in the proximal caput 28 days after thymidine injection. After 2 d labeled sperm had arrived at the distal caput and, after another 2, the bulk of labeled sperm was found in the corpus. From there the sperm advanced to the transition of corpus and cauda, where progress was arrested until Day11. On Day 12, transit was resumed and by Day 13 sperm had passed through the cauda and vacated the epididymis via the ductus deferens.

Vitrification of Mouse Blastocysts by Open or Closed System and Warming in Sucrose-containing or Sucrose-free Diluent.

BACKGROUND: Cryopreservation of embryos is of considerable relevance for the implementation of embryo transfer programs and the establishment of embryo banks in several mammalian species. OBJECTIVE: The present investigation compares two different vitrification systems and two different warming solutions. MATERIALS AND METHODS: Vitrification was performed using Open Pulled Straw (OPS) or CVM RingFibre plug (CVM) devices. Warming was carried out either in a warming solution containing 0.33 M sucrose or in a solution devoid of sucrose. RESULTS: Differences between vitrification systems were not significant. Warming in sucrose-containing diluent resulted in an expansion rate of 64%, as compared to 86% in a solution devoid of sucrose; reported hatching rates were 45% vs. 9%, respectively (p<0.05). Upon transfer, implantation rates for OPS- and CVM were 50% and 27%, respectively, compared with 55% for freshly collected embryos. The implantation rate after warming was 43% for sucrose-containing and 33% for sucrose-free medium. CONCLUSION: a) both vitrification systems are suitable for vitrifying mouse blastocysts; b) warming in sucrose-free diluent yields better embryo survival rates than in diluent containing 0.33 M sucrose.

Comparison of various semen extenders and addition of prostaglandin F2α on pregnancy rate in cows.

This investigation comprises three trials. Trial 1 consists of an in vitro comparison of three semen extenders: two egg yolk based (customized Tris-egg yolk-glycerol and Triladyl®), the third (AndroMed®) soybean lecithin based. With regard to post-thaw motility, the phytoextender AndroMed® proved to be superior (59±3% v. 53±2% and 53±2%, P<0.05). It had earlier been shown that addition of the commercial prostaglandin F2α preparation Dinolytic® before freezing compromises post-thaw motility; therefore, in Trial 2, Dinolytic® was added after thawing. Frozen-thawed spermatozoa tolerated addition of Dinolytic® at a concentration of 30% (v/v). In Trial 3, cows were inseminated using straws in which diluted semen and Dinolytic® were frozen in the same straw, separated by an air bubble, so intermingling could only take place in the course of insemination. Pregnancy rates at Dinolytic® dosages of 0%, 30% or 60% amounted to 44%, 41% and 56%, respectively (P>0.05), a result that encourages a large-scale field study, which is envisioned.

Fixed-time Insemination in Pasture-based Medium-sized Dairy Operations of Northern Germany and an Attempt to Replace GnRH by hCG.

A field study was conducted aimed at (i) evaluating the practicability of a fixed-time insemination regime for medium-sized dairy operations of north-western Germany, representative for many regions of Central Europe and (ii) substituting hCG for GnRH as ovulation-inducing agent at the end of a presynch or ovsynch protocol in an attempt to reduce the incidence of premature luteal regression. Cows of two herds synchronized by presynch and two herds synchronized by ovsynch protocol were randomly allotted to three subgroups; in one group ovulation was induced by the GnRH analog buserelin, in another by hCG, whereas a third group remained untreated. The synchronized groups were fixed-time inseminated; the untreated group bred to observed oestrus. Relative to untreated herd mates, pregnancy rate in cows subjected to a presynch protocol with buserelin as ovulation-inducing agent was 74%; for hCG it was 60%. In cows subjected to an ovsynch protocol, the corresponding relative pregnancy rates reached 138% in the case of buserelin and 95% in the case of hCG. Average service interval was shortened by 1 week in the presynch and delayed by 2 weeks in the ovsynch group. It may be concluded that fixed-time insemination of cows synchronized via ovsynch protocol with buserelin as ovulation-inducing agent is practicable and may help improve efficiency and reduce the work load involved with herd management in medium-sized dairy operations. The substitution of hCG for buserelin was found to be not advisable.

TEMPORARY STORAGE OF BOVINE SEMEN CRYOPRESERVED IN LIQUID NITROGEN ON DRY ICE AND REFREEZING OF FROZEN-THAWED SEMEN.

Two experiments were conducted. The purpose of Experiment 1 was to investigate whether viability of bovine semen stored in liquid nitrogen (-196°C) will be adversely affected by temporary exposure to dry ice (-79°C). It was convincingly shown that post thaw-motility was not affected, regardless whether semen was thawed immediately or after being returned to liquid nitrogen. Shipping or temporary storage on dry ice, thus, is a viable option. In Experiment 2, refreezing of frozen-thawed semen was attempted. The proportion of motile spermatozoa was reduced by a factor of ten to between 6.0 % and 7.4 %, regardless whether thawing occurred directly after removal from liquid nitrogen or after an interim period on dry ice. When semen was refrozen on dry ice before being returned to liquid nitrogen, motility rates were significantly improved (13.0 % to 17.0 %, P<0.05). In both experiments sperm cells that remained motile displayed vigorous forward movement and normal morphological appearance.

Carcass Characteristics and Meat Quality of Swamp Buffaloes (Bubalus bubalis) Fattened at Different Feeding Intensities.

Twenty-four male 1-year old swamp buffaloes (Bubalus bubalis) were randomly allocated to 4 groups. One group grazed on guinea grass (GG) and another on guinea grass and the legume Stylosanthes guianensis (GL). The other two groups were kept in pens and fed freshly cut guinea grass and concentrate at an amount of 1.5% (GC1.5) and 2.0% (GC2.0) of body weight, respectively. The effect of the different feeding intensities on carcass characteristics and meat quality were assessed. The mean body weight at slaughter was 398 (±16) kg. Average daily gain was higher in concentrate-supplemented groups (570 and 540 g/d in GC1.5 and GC2.0, respectively) when compared to GG (316 g/d) and GL (354 g/d) (p<0.01). Likewise, the warm carcass weight was higher in GC1.5 and GC2.0 compared to GG and GL. Dressing percentage was 48.1% and 49.5% in GC1.5 and GC2.0 in comparison to 42.9% and 44.8% observed in GG and GL, respectively. Meat of Longissimus throracis from GC1.5 and GC2.0 was redder in color (p<0.01), while water holding capacity (drip and thawing loss) was improved in pasture-fed groups (p<0.05). Protein and fat content of Longissimus thoracis was higher in animals supplemented with concentrate (p<0.01), as was cholesterol content (p<0.05), whereas PUFA:SFA ratio was higher and n-6/n-3 ratio lower (p<0.01) in pasture-fed buffaloes. Results of the present study showed that the supplementation of pasture with concentrate enhances the growth and carcass characteristics of swamp buffaloes expressed in superior dressing percentage, better muscling, and redder meat with a higher content of protein and fat, whereas animals grazing only on pasture had a more favorable fatty acid profile and water holding capacity. In conclusion, the supplementation of concentrate at a rate of about 1.5% of body weight is recommended to improve the performance and carcass quality of buffaloes.

Pregnancy-associated glycoprotein (PAG) profile of Holstein-Friesian cows as compared to dual-purpose and beef cows.

Pregnancy-associated glycoproteins (PAGs) are produced by mono- and binucleate trophoblast cells in the ruminant placenta. PAG appears in maternal blood and, from approximately 4 weeks after fertilization onward, may serve as a reliable means of diagnosing pregnancy. A range of factors are said to affect plasma PAG concentrations, such as number and sex of foetus, mass of calf and placenta, level of milk production and genetic constitution. In this study, PAG pregnancy profiles of a dual-purpose (Simmental) and two beef breeds (Uckermark and Aubrac) are compared with the profile of the specialized dairy breed Holstein-Friesian. Holstein-Friesian cows were sampled weekly; the levels of the other breeds were presented at 3-week intervals. The overall significant breed difference (p = 0.013) was founded on deviations during the initial 3 weeks of pregnancy and from 23 weeks onward. During the period critical for the detection of pregnancy, between four and 22 weeks, agreement between PAG levels of various breeds was close (p > 0.05). No significant effect of body mass of cow or calf (relative to mass of dam) was detected. These findings imply that the PAG pregnancy test may be executed uniformly irrespective of breed or type of cow, affirming the suitability of the test as a valuable asset for the cattle industry.

Short communication: a note on the correction for the effect of freezing on the outcome of pregnancy-associated glycoprotein measurement in blood and serum of cows.

Early pregnancy detection is a measure of considerable economic relevance for dairy cattle breeders, and analysis of pregnancy-associated glycoprotein (PAG) values in blood is one of the methods implemented in practice. Starting from d 30 postconception, cows are considered to be pregnant at PAG levels of 2.0 ng of PAG/mL of blood and higher. However, little is known about preanalytic sources of errors that might affect PAG values. Based on blood samples from 65 dairy cows, the present study showed that freezing of samples, such as may be the case during shipping in wintertime, will lower PAG values considerably. Therefore, a Bland-Altman analysis was used to derive a correction factor. Overall, the mean differences (± standard deviation) between frozen and respective fresh samples was -5.5 ± 7.4 ng of PAG/mL of blood and 0.9 ± 6.1 ng of PAG/mL of serum. However, the Bland-Altman plot revealed a concentration-dependent effect of freezing on PAG values with higher variability and larger declines at higher PAG levels. Therefore, to minimize chances of false-negative results, different correction factors are suggested for different levels of PAG (e.g., based on the upper bound of the 95% confidence interval 0.67 for PAG levels between 2.0 and 3.9 ng of PAG/mL and 0.25 for PAG levels between 4.0 and 7.9 ng of PAG/mL). With these concentration-dependent correction factors, implementation into practice will be possible. The accuracy is adequate because no quantitative information but qualitative results (pregnant vs. nonpregnant) are required. However, due to larger chances of false-negative results, the application of the correction factor should only be a last resort if temperature exposure of a sample is unknown.

Is sucrose required in open pulled straw (OPS) vitrification of mouse embryos?

The study addresses the relevance of sucrose with OPS vitrification of murine blastocysts. In a 3 x 3 factorial experiment, blastocysts were subjected to vitrification solution (20% Me2SO and 20% EG) containing 0.0, 0.4 or 0.8M sucrose and warming solution (HEPES buffered TCM 199) containing 0.00, 0.25 or 0.50M sucrose. After 48h of in vitro culture, with 0.4 M sucrose in vitrification solution, 84-87% of embryos had reached the expanded blastocyst stage, as compared to 76-82% with 0.0M and 40-54% (P < 0.01) with 0.8M sucrose. Hatching rates confirmed the tendency. The sucrose content of the warming solution had no significant effect on expansion or hatching rates (P > 0.05). It may be concluded that, whereas, vitrification solution should contain a moderate concentration of sucrose, in dilution medium sucrose is dispensable. This implies that embryos may be transferred directly after warming, which, if applicable to farm animals, would greatly facilitate vitrification in practice.

The effect of ph on the fertilizability of rainbow trout (Oncorhynchus mykiss) eggs stored in a chilled state.

To verify the observation that fertilizability of stored rainbow trout eggs is affected by deviations in pH (Komrakova and Holtz, 2011) freshly collected eggs were immersed in coelomic fluid adjusted to pH 6, 7, 8, 9, or 10 (3 replicates each) and stored at 2 degreesC. At 0, 1, 24, 48, and 96h, pH was measured and eggs were inseminated with cryopreserved semen. Egg development until the eyed stage proceeded normally in all cases. However hatching rates, amounting to 70% in controls (pH 8, SEM 0.02), were reduced to 50% in samples exposed to pH 7 or pH 9 and to 40% and 30% in samples exposed to pH 6 and pH 10, respectively. In conclusion, when eggs are subjected to coelomic fluid with deviating pH, the damage afflicted upon the eggs will not become evident until they are due to hatch.

Pharmacokinetics of human chorionic gonadotropin after i.m. administration in goats (Capra hircus).

The present investigation addresses the pharmacokinetics of human chorionic gonadotropin (hCG), intramuscularly (i.m.) administered to goats. Nine pluriparous does of the Boer goat breed, 2-6 years of age and weighing 45-60 kg, were administered 500 IU hCG (2 ml Chorulon) deep into the thigh musculature 18 h after superovulatory FSH treatment. Blood samples were drawn from the jugular vein at 2  h intervals for the first 24h, at 6 h intervals until 42 h, and at 12 h intervals until 114 h after administration. After centrifugation, plasma hCG concentrations were determined by electrochemiluminescence immunoassay. Pharmacokinetical parameters were as follows: lag time, 0.4 (s.e.m. 0.1) h; absorption rate constant, 0.34 (s.e.m. 0.002) h; absorption half-life, 2.7 (s.e.m. 0.5) h; elimination rate constant, 0.02 (s.e.m. 0.002) h; biological half-life, 39.4 (s.e.m. 5.1) h; and apparent volume of distribution, 16.9 (s.e.m. 4.3) l. The plasma hCG profile was characterized by an absorption phase of 11.6 (s.e.m. 1.8) h and an elimination phase of 70.0 (s.e.m. 9.8) h, with considerable individual variation in bioavailability and pharmacokinetical parameters. Biological half-life was negatively correlated (P<0.05) with peak concentration (r=-0.76), absorption rate constant (r=-0.78), and elimination rate constant (r=-0.87). The results indicate that after rapid absorption, hCG remains in the circulation for an extended period. This has to be taken into account when assessing the stimulatory response to hCG treatment on an ovarian level.

The effect of exogenous gonadotropins on ovarian function in goats actively immunized against inhibin.

The aim of this investigation was to compare the ovarian response to superovulatory treatments in does before and after inhibin immunization, with a view to optimizing the superovulatory potential of the caprine ovary. To avoid interference by the ovarian cycle, the experiment was conducted out-of-season. At the onset of the experiment 48 does were subjected to treatment with an sc implant of the progestogen norgestomet, combined with a gonadotropin; eight does each received a single injection of 1200 IU eCG, 400 IU eCG or 2 mL physiological saline (control) or six injections (at 12 h intervals) constituting 16 or 5.4 AU pFSH. The does were mated and subjected to embryo collection 6 to 7 d later. Throughout the experiment ovarian function (by ultrasonography) and plasma levels of inhibin antibodies and progesterone were monitored. Of 40 does treated during the first part of the experiment, 48% showed estrus. The ovarian response in does treated with a high or low dose of eCG or a low dose of pFSH was barely in excess of the ovarian response in the saline-treated controls, whereas a superovulatory dose of pFSH (16 AU) gave a satisfactory response of, on average, 14.5 ovulations (yielding 8.8 flushed ova and embryos). Immediately after the does had been subjected to embryo collection they were actively immunized against inhibin by administering two injections of a recombinant α-subunit of ovine inhibin at four week intervals. All immunized does produced antibodies with the maximal titer reached two weeks after the second injection. Groups of immunized does were subjected to the same gonadotropin treatments as before (avoiding allocation of individuals to the same treatments). This time all does showed estrous symptoms. The ovulatory response to the various treatments, including the saline controls, was virtually identical, the overall average being 21.8 follicles and 9.1 ovulations. The average embryo yield per doe was 5.7. The results imply that inhibin acted as the key factor in determining the ovulatory response since no impact of any of the supplementary gonadotropins was noted in inhibin-immunized does. This finding gives rise to the notion that inhibin antibodies may act primarily by an intraovarian paracrine action rather than by reducing the suppressive action of inhibin on pituitary FSH release. Further, these findings confirm earlier reports that eCG is less suitable than FSH for inducing superovulation in goats, and indicate that active immunization against inhibin may be considered a viable alternative to using exogenous gonadotropin for inducing superovulation in goats.

Fixed-time deep uterine insemination in PGF2α-synchronized goats.

The aim of this investigation was to optimize fixed-time insemination in goats by clustering ovulations in prostaglandin F(2α)-synchronized goats either with gonadotropin releasing hormone (GnRH) or human chorionic gonadotropin (hCG). The underlying intention was to reduce the incidence of short cycles by providing a more sustained stimulation of the corpus luteum by substituting the commonly used GnRH with longer-acting hCG. It was conjectured that this might render the corpus luteum less prone to premature regression. Sixty pluriparous does were administered 5 mg of the prostaglandin F(2α) preparation dinoprost (Dinolytic; Pharmacia and Upjohn, Erlangen, Germany) during the luteal phase of the estrous cycle. Twenty of these does were administered 0.004 mg of the GnRH analog buserelin (Receptal; Intervet, Unterschleissheim, Germany) 48 hours later; another 20 does received 500 IU hCG (Chorulon; Intervet, Unterschleissheim, Germany) instead. Sixteen hours later the does were inseminated with frozen-thawed semen. The remaining 20 does served as controls and were inseminated 16-18 h after the onset of detected estrus. All 60 treated goats displayed estrous symptoms, the time of onset being similar for all groups (42.6, 37.6, and 40.5 hours after treatment for GnRH-treated, hCG-treated, and control does, respectively). The duration of estrus in the GnRH-treated group was 10 h less than in the other groups (45.1 vs. 56.4 and 54.4 h, P < 0.05). The number of ovulations (assessed by ultrasound monitoring) did not differ among groups (2.4, 2.1, and 2.5, P > 0.05). Monitoring of serum progesterone revealed that the incidence of corpus luteum insufficiency was significantly higher in GnRH- and hCG-treated does than in the control group (40% and 35% vs. 5%, P < 0.05). The pregnancy rate was 50% in the GnRH and 35% in the hCG group as compared with 60% in the controls. Corresponding kidding rates were 40%, 35%, and 60% (P > 0.05). When disregarding does with corpus luteum insufficiency, pregnancy rates would have been 83%, 54%, and 63%, and kidding rates 67%, 54%, and 63%, respectively. The average number of kids born was 1.88, 1.71, and 1.83, respectively (P > 0.05). It may be concluded that fixed time insemination of cycling does treated with prostaglandin F(2α) during the luteal phase, followed by ovulation induction with GnRH or hCG, would be an effective management tool if it were possible to control the high incidence of corpus luteum insufficiency. The attempt to achieve this by substituting GnRH with hCG, was not met with success. Until a solution for the problem has been found, it is advisable to inseminate prostaglandin-synchronized does 16-18 hours after the onset of detected estrus.

The effect of prostaglandin F(2α) administration at the time of insemination on the pregnancy rate of dairy cows.

This paper addresses the question whether the pregnancy rate of dairy cows and heifers may be affected by administering prostaglandin F(2α) at the time of artificial insemination. A field trial involving 1031 dairy cows and heifers distributed to a large number of small dairy farms in an area of extensive farming in central Germany provided evidence that intramuscular administration of 25mg Dinoprost (Dinolytic(®)) at the time of insemination has no effect on pregnancy rate (61% of the cows and heifers were pregnant in both prostaglandin F(2α)-treated and saline control groups). On the other hand, deposition of 0.5mL of a 0.5mg/mL Dinoprost solution in the uterine lumen immediately after artificial insemination gave rise to a pregnancy rate of 66% as compared with 59% in saline controls. The increase in pregnancy rate of 229 prostaglandin F(2α)-treated animals (66% pregnant) over that of 226 saline controls (59% pregnant) amounted to 12%. This improvement was not statistically significant (P=0.12). Factors exerting a significant effect on pregnancy rate were parity (74% pregnancies in heifers versus 57% in cows, P<0.01 and 65% pregnancies in first parity-cows versus 55% in older cows, P<0.01) and season (57% during the barn season versus 64% during the pasture season, P<0.05), whereas length of service period, level of milk production and serum or milk progesterone level at the time of insemination did not. A follow-up trial involving more animals will have to be conducted aimed at confirming the promising results obtained by intrauterine PGF(2α) administration.

Open pulled straw vitrification of goat embryos at various stages of development.

This investigation addresses the question whether it is possible to apply the open pulled straw (OPS) vitrification method, found to be effective for cryopreserving caprine (Capra aegagrus hircus) blastocysts, to other embryonal stages. Morulae, blastocysts and hatched blastocysts were cryopreserved by way of OPS vitrification and blastocysts and hatched blastocysts by conventional freezing. Morulae were not included with conventional freezing because in our experience the survival rate is very low. To assess the viability of the cryopreserved embryos, they were transferred to synchronized does; in most cases, two embryos per doe. After OPS vitrification, of nine does receiving morulae, not a single one became pregnant; of 11 does receiving blastocysts, nine (82%) became pregnant (all of which kidded and gave birth to, on average, 1.8 kids); and of nine does receiving hatched blastocysts, three (33%) became pregnant (two of which [22%] kidded, giving birth to a single kid each). After conventional freezing, of 10 does receiving blastocysts, five became pregnant (four of which [40%] carried to term and gave birth to a pair of twins each); and of nine does receiving hatched blastocysts, three (33%) became pregnant (and gave birth to a single kid each). Embryo survival (kids born/embryos transferred) after vitrification for morulae, blastocysts, and hatched blastocysts was 0, 70% (16 of 23), and 13% (2 of 16), respectively, and after conventional freezing for blastocysts and hatched blastocysts was 42% (8 of 19) and 19% (3 of 16), respectively. The difference in pregnancy and kidding rate between vitrified and conventionally frozen blastocysts was significant, and so was the difference in pregnancy rate between hatched and nonhatched blastocysts, regardless whether OPS-vitrified or conventionally frozen. The results of the current study indicate that OPS vitrification is a very effective means of cryopreserving caprine blastocysts. Unfortunately, the superiority of OPS vitrification over conventional freezing does not apply to caprine morulae and hatched blastocysts.

In vitro and in vivo survival of mouse blastocysts after repeated vitrification with the open pulled straw (OPS) method.

This study addresses the in vitro and in vivo survival of mouse embryos repeatedly vitrified by the OPS-technique of Vajta et al. (24). Of 225 vitrified blastocysts, after warming one third was cultured in vitro, the other two-thirds were re-vitrified. After these were warmed, the second third was put to culture. With the remaining third the procedure was repeated. Of embryos vitrified once, 97% developed to expanded blastocysts and 81% proceeded to hatching. Corresponding values for embryos re-vitrified once were 93% and 72%, respectively (P > 0.05). After another re-vitrification, expansion and hatching rates were reduced to 76% and 35%, respectively (P < 0.01). Of 10 recipients provided with 10 embryos each that had been vitrified once, 80% remained pregnant with 5.5 +/- 0.3 fetuses. Corresponding values for re-vitrified embryos were 80% and 5.0 +/- 0.3. Of all embryos transferred, 44% became vital fetuses after a single vitrification, compared to 40% after revitrification.

Establishment of an ELISA for measuring bovine pregnancy-associated glycoprotein in serum or milk and its application for early pregnancy detection.

It has been shown in several mammalian species that during pregnancy, trophoblast cells express a range of pregnancy-associated glycoproteins (PAG). The presence of PAG in the maternal serum of cows may serve as an indicator of pregnancy from day 28 after AI onward. The present study addresses (1) conversion of an existing PAG-RIA to a competitive double antibody ELISA using a polyclonal anti-bPAG-IgG and an anti-rabbit-IgG raised in sheep for coating and (2) application of newly established ELISA to test its suitability for pregnancy detection by measuring PAG in serum or milk. The intraassay coefficients of variation (CV) for the PAG-ELISA were 2-14% for serum and 10-12% for milk; the corresponding interassay CVs were 8-22% and 12-22%, respectively. Pregnancy-associated glycoprotein profiles established in milk and serum of 12 pregnant cows showed a characteristic pattern with measurable amounts from approximately day 20 onwards in serum and from day 60 onwards in milk. In a field trial, serum PAG was determined in 397 cows sampled between 20 and 50 days after insemination. The outcome was that, pregnancy could reliably be diagnosed from day 28 onwards in serum and from day 150 onwards in milk. In conclusion, it may be stated that the established ELISA provides an efficient and reliable means of pregnancy diagnosis that will, in our judgement, gain in popularity with cattle breeding. The ELISA proved to be an adequate and efficient way of measuring PAG in maternal serum or milk and will be a useful means of pregnancy detection in cows.

Coating of objects introduced into the oviduct of pseudopregnant rabbit does.

This investigation addresses the possibility of providing mouse embryos or other foreign objects with a protective mucin coat by transferring them into the oviduct of a live rabbit doe. Mouse embryos at the 8 or 16-cell stage, rabbit oocytes and latex spheres resembling mouse embryos in size were transferred to the ligated oviducts of ovulation-induced rabbit does. The does were killed 24 h later to have their oviducts flushed. A large proportion of the latex spheres (89%) and of the ovulated oocytes of the recipient does (92%) was recovered. The recovery rates for transferred rabbit oocytes, either intact or with the zona pellucida removed, were 61% and 51%, respectively, whereas that for mouse embryos was extremely poor (20%). Rabbit oocytes with or without zona were enveloped in a thick mucin coat regardless whether they had been transferred or ovulated by the recipients. The same applied to empty rabbit zonae. Mouse embryos and latex spheres were also covered by a mucin coat, but it was four times thinner. While residing in the rabbit oviduct, the mouse embryos continued developing to a stage comparable to what would have been expected in situ. During the subsequent in vitro culture, mouse embryos continued developing to the expanded blastocyst stage. They did, yet, not hatch from the zona. It may be concluded that particles of various origins, when placed into the oviduct of ovulated rabbit does, will be provided with a mucin covering which is, however, considerably thinner than that surrounding oocytes or zonae pellucidae originating from rabbits.

The long-term effect of active immunization against inhibin in goats.

This experiment addresses the long-term effect of active immunization of goats against a recombinant ovine inhibin alpha subunit (roIHN-alpha). In late anestrus 100microg of roINH-alpha was administered to 40 pluriparous Boer goat does, followed, 4 weeks later, by a booster injection. Weekly blood samples were drawn to monitor the inhibin binding capacity with the aid of a radio-tracer binding assay. From the onset until 48h after the end of each estrus, follicular development and ovulation rate were monitored at 24h intervals by transrectal ultrasonography. Beginning in August and continuing into January, does were mated at every other estrus, and submitted to transcervical embryo collection. Seven months after the first immunization, the does were mated again and permitted to carry to term. All immunized does produced inhibin antibodies, an elevated titre being first detected 2 weeks after primary immunization. Maximum titres were reached after 6 weeks, i.e. 2 weeks after the booster injection. Thereafter, in the course of the following 32 weeks, the titre subsided gradually. The does started cycling by mid-August. At that stage the average number of follicles more than 4mm in diameter, ovulations and total embryos and ova recovered were 14.7 (+/-2.3), 5.3 (+/-0.7) and 4.4 (+/-1.0), respectively. A steady decline followed and in January the corresponding means were: 5.2 (+/-0.6) follicles, 3.1 (+/-0.6) ovulations and 1.2 (+/-0.4) embryos and ova recovered. When mated toward the end of the breeding season, 85% of the does became pregnant to the first mating and 73% went to term. Healthy kids were born, the average litter size being 2.2 (+/-0.1). In conclusion, immunization of goats against a recombinant inhibin alpha-subunit proved to be a practicable means of producing embryos for transfer purposes. After about half a year, when the inhibin antibody titre has subsided, it is possible to return the does to the breeding flock without risking complications with normal breeding activity.