Pregnancy stage and number of fetuses may influence maternal plasma leptin in ewes.

Maternal plasma leptin is elevated in ewes during pregnancy. The authors studied whether there was any relation between maternal plasma leptin and insulin concentrations, the number of fetuses and the circulating and faecal levels of gestagens. At the end of the breeding season in January the ovarian activity of Prolific Merino ewes was induced/synchronised with gestagen + eCG treatment. Ewes were inseminated artificially (AI) by laparoscopy. Blood and faecal samples were collected before AI (day 0) and again 41, 81 and 101 days later. The plasma levels of leptin (pL), insulin and progesterone (pP4), and the faecal P4 metabolite (P4-met) content were determined. The day 0 level of pL was significantly higher in pregnant (n = 24) than in non-pregnant ewes (n = 32). By day 41 the pL of pregnant animals had doubled, it showed a further moderate increase on day 81, and decreased slightly thereafter. During pregnancy pP4 and faecal P4-met rose continuously and were positively correlated at all stages. The mean levels of pL and pP4 and the faecal content of P4-met were lower in ewes bearing single (n = 12) than in those with 2 (n = 6) or 3-5 fetuses (n = 6). Analysis of variance demonstrated significant differences according to the number of fetuses in the pL and pP4, but not in P4-met (p = 0.042, 0.044, and 0.051, respectively). Leptin showed positive correlation with insulin before the AI but not during pregnancy. On days 41 and 81 pL showed a slight positive correlation with P4 and P4-met, which decreased slightly by day 101. This study shows that although leptinaemia is affected by the number of fetuses and the level of P4, pregnancy stage is a more important regulator than these additional factors.

Development of a sensitive ELISA for the determination of fumonisin B(1) in cereals.

Monoclonal fumonisin B(1) antibodies with high titer were raised by using FB(1)-glutaraldehyde-keyhole limpet hemocyanin immunogen prepared by a short cross-linker reagent (glutaraldehyde). Mean cross-reactivities of the selected monoclonal antibody for FB(1), FB(2), and FB(3) were 100, 91.8, and 209%, respectively; no reactivity was found with hydrolyzed fumonisin. A direct, competitive enzyme-linked immunosorbent assay for the quantitative determination of FB(1) in cereals has been developed with this antibody. Fifty percent acetonitrile-based solvent with some additives was used for extraction of cereals, and the diluted extracts were used without cleanup in the test. The mean within-assay and interassay coefficients of variation for the standard curve were <10%. The measuring range of this test is 10-500 ng/g, with a detection limit of 7.6 ng/g FB(1). The toxin recovery from cereals infected with 50-200 ng/g of FB(1) varied between 61 and 84%. According to the comparable results of naturally infected maize samples, this test proved to be suitable for the rapid screening of food and feed samples for the presence of FBs.

Ochratoxin A content of human sera determined by a sensitive ELISA.

A sensitive, monoclonal antibody-based ELISA test was developed and used for quantitative determination of ochratoxin A (OA) in human sera. The measuring range of this test (without sample dilution) was 0.2-2.0 ng/mL, and the detection limit was 0.2 ng/mL. The OA concentrations of 355 sera samples varied from < 0.2 to 10 ng/mL OA, but 75% of the samples contained 0.2-1.0 ng/mL. This amount reflects a tolerable daily intake (TDI) value of toxin. However, in some cases (6.8%), more than 1.0 ng/mL OA was measured, which is probably a result of elevated intake of OA, which may even exceed the "virtually safe dose". Our data indicate that, like in many other countries, OA is present in food or feed products available in Hungary, and in order to save the health of consumers, their regular control is desirable.

Analysis of serum and seminal plasma after feeding ochratoxin A with breeding boars.

The aim of the experiment was to investigate whether or not ochratoxin A (OA) can be detected in seminal plasma after feeding the toxin in five and 10 times of the human tolerable daily intake with breeding boars and how toxin profiles of serum and seminal plasma correspond to each other. In addition to that, the effect of the toxin challenge on motility and longevity of boar semen was also evaluated. OA from samples was analyzed by microplate ELISA. Percentage of progressive motility of spermatozoa was determined initially and after 24, 48, 96, 120 and 144 h of storage. OA appeared in serum and seminal plasma shortly after toxin application had started. Significant reduction of initial motility and impaired longevity was observed after toxin withdrawal. These findings suggest that OA might have the potential to affect sperm production and semen quality of boars, but further research is required to elucidate whether OA exerts direct effect on germinal epithelium or disturbs sperm cell maturation only.

Influence of beta-carotene on fertility in rabbits when using embryo transfer programs.

The effect of beta-carotene on reproduction traits in rabbits was studied in 509 (superovulated and normally ovulated) donors and 239 recipients by using embryo/gene transfer performed at 2 different locations. All of the bucks and the half of the females were fed a diet supplemented with 40 mg synthetic beta-carotene (Rovimix((R)))/kg feed. Embryos at the pronucleus stage were collected 19 to 21 hours after induction of ovulation with human chorionic gonadotropin (hCG); they were then microinjected into the male pronucleus and transferred to synchronized recipients. Data were obtained from the time when the donors and recipients were caged, until the pups resulting from the embryo transfers were weaned. Supplemented beta-carotene did not affect most of the 30 traits that were analyzed. However superovulated donors in Project 2 that received the beta-carotene enriched diet had a 14% lighter ovary weight (P<0.05) and less than half of the oocytes were unfertilized (P<0.05). In Project 1 (beta-carotene group) there was a greater number of pups born (36%, P<0.05) and more of these pups were born alive (53%, P<0.05).

Economical and ecological importance of indigenous livestock and the application of assisted reroduction to their preservation.

Among the many mammalian species that are threatened as the result of habitat destruction are numerous species of rare or little-known native livestock that possess features that render them ideally adapted to their environment. Because of the vital and valuable role many of these species play both to the ecology and economy of their native countries, attention is being directed towards initiating breeding programs that might insure their continued survival. This review introduces and highlights the importance of some of these indigenous species and outlines efforts currently underway to apply assisted reproductive technologies to their conservation.

Ovarian response, embryo recovery and results of embryo transfer in a Hungarian native pig breed.

The objective of the study was to use embryo transfer (ET) for propagation of the Swallow Belly Mangalica population. Mangalica is a native Hungarian pig breed adapted to extreme climate and housing conditions and distinguished for excellent meat and fat quality. However, due to their weak reproductive characteristics and relatively high fat proportion, Mangalica pigs have been replaced by modern breeds. Now, there is an increased interest again to safeguard the properties of this breed. We conducted two experiments. First, we used a total of 18 puberal Mangalica gilts to determine an optimal superovulatory treatment. Following estrus synchronization with Regumate, we injected gilts with either 750, 1000 or 1250 IU PMSG, followed by 750 IU hCG 80 h later. We scanned ovaries endoscopically 3 days after hCG administration. The application of 1000 and 1250 IU PMSG resulted in a higher rate of ovulation compared to 750 IU (24.2 +/- 3.6 and 21.0 +/- 2.3 vs. 13.7 +/- 2.7 P<0.05). The number of follicular cysts increased after administration of 1250 IU PMSG compared to 750 and 1000 IU (2.0 +/- 1.3 vs. 0.3 +/- 0.7 and 0.2 +/- 0.3, P<0.05). Thus, we chose 1000 IU PMSG for further stimulation of Mangalica gilts. In the second experiment, we induced superovulation in 10 Mangalica donor gilts by 1000 IU PMSG and 750 IU hCG. Gilts were fixed-time inseminated, and then five days later embryo collection was carried out surgically (n=6) or endoscopically (n=4). Out of the 187 ova recovered, 92.5% were at the morula/blastocyst stage. The embryo recovery rate was higher following surgical flushing than following endoscopy (91.5 +/- 4.4% vs. 71.4 +/- 12.7%, P<0.05). Altogether 143 embryos were transferred surgically or endoscopically into 8 Landrace recipients. Surgical and endoscopic transfer of Mangalica embryos into Landrace gilts resulted in pregnancies in 3 and 2 gilts, respectively; thus the overall farrowing rate was 62.5%. The birth of 59 Mangalica piglets from 5 embryo recipients equals an average litter size of 11.8 +/- 1.3, which is two times larger than usual in this breed. Therefore, we concluded that an appropriate inter-breed ET program is a suitable tool to propagate the endangered Mangalica breed.

Factors influencing ovarian activity and sexual behavior of postpartum mares under farm conditions.

Management of the postpartum period is one of the most important factors of stud farm medicine. In horses, owing to the long gestation period, the time from parturition to repeat conception needs be short to maintain an optimal yearly foaling interval. For this reason the features of postpartum ovarian activity and sexual behavior were studied under farm conditions. During 2 consecutive breeding seasons, 107 mares on 5 commercial horse farms were monitored after parturition by regular teasing, transrectal ultrasonography and blood sampling for progesterone. Foalings took place from January 1 to June 15. Body condition scoring was carried out within 5 d and at 60 to 65 d after parturition. The first ovulation occurred within 20 d after foaling in 84.1% (90/107) of the mares. The mean intervals from foaling to the first and second ovulations were 17.8 +/- 1.6 d (+/- SEM) and 40.9 +/- 2.7 d (+/- SEM), respectively. The mean intervals from parturition to the first and second ovulation (P < 0.001), the interovulatory interval (P < 0.01), the second follicular phase (P < 0.001), and the time until the first overt estrus (P < 0.01) were significantly longer in mares foaling before the vernal equinox. In the beginning of the breeding season the intervals from parturition to the first ovulation (P < 0.01), to the second ovulation (P < 0.01), and to the first overt estrus (P < 0.001) were significantly longer for primiparous mares than for multiparous animals. There was a tendency for an increased interovulatory interval and for a longer second follicular phase in mares with decreased body condition after parturition (P = 0.069, P = 0.089, respectively). Suckling and breed had no effect on postpartum ovarian activity. We concluded that under field conditions the resumption of cyclic ovarian activity and sexual behavior in mares after foaling are strongly affected by the season of parturition and parity. In some cases, body condition change and other factors may also play a role in influencing postpartum reproductive function.

Vitrification of mouse embryos in two cryoprotectant solutions.

The objective of this study was to compare the efficiency of 2 media on the vitrification of mouse compacted morulae, early blastocysts and expanded blastocysts after equilibration at room temperature of 4 degrees C. Embryos were equilibrated for 10 min in either 25% VS3 (Rall Equilibration Medium, REM) or 10% glycerol + 20% propylene glycol (Massip Equilibration Medium, MEM) in DPBS at 20 degrees C or 4 degrees C. For vitrification either 100% VS3 (Rall Vitrification Medium, RVM) or 25% glycerol + 25% propylene glycol (Massip Vitrification Medium, MVM) in DPBS was used. Embryos equilibrated at room temperature were loaded in 20 microL of vitrification media into 250 microL straws and then immediately (30 sec) plunged into liquid nitrogen (LN2). After equilibration at 4 degrees C the embryos were put into straws with 20 microL of precooled vitrification medium, and after 20 min at 4 degrees C they were plunged into LN2. Embryos from both groups were thawed in a 20 degrees C water bath for 20 sec, transferred to 1.0 M sucrose in DPBS for 5 min and then cultured for 24 to 48 h in Whitten's medium at 37 degrees C in 5% CO2 in air. In the groups of embryos prepared for vitrification at room temperature the survival rate of compact morulae vitrified in RVM was higher than those vitrified in MVM (65/70, 93% vs 49/74, 66%; P < 0.01). No difference was found in the survival rate of early blastocysts and expanded blastocysts vitrified in RVM or MVM (30/83, 36% vs 25/75, 33% and 4/66, 6% vs 4/76, 5%). No difference was found between the survival rate of compact morulae after equilibration with RVM or MVM at 4 degrees C (62/75, 83% vs 52/74, 70%). Both the early blastocysts and expanded blastocysts equilibrated at 4 degrees C MVM yielded a higher survival rate than RVM (28/74, 38% and 40/70, 57% vs 4/75, 5% and 4/77, 5%; P < 0.01). We conclude that, of the 3 developmental stages, compact morulae withstand the vitrification process best, and reduction of the temperature prior to plunging into LN2 is not required. A 10-fold increase in the survival rate of expanded blastocysts can be achieved using low temperature equilibration (4 degrees C) and MVM.

Latent immunization to produce high-affinity monoclonal antibodies to progesterone.

A simple immunization method to obtain high-affinity monoclonal antibodies to progesterone is described in this article. The method is based on the theory of affinity maturation. A long interval between antigen priming and booster ("latent immunization") permits an undisturbed completion of affinity maturation, resulting in the accumulation of memory B lymphocytes with high-affinity Ig receptors, and consequently, in a higher rate of hybridoma clones producing high-affinity antibody after cell fusion. Antibodies obtained after hyperimmunization and latent immunization are compared in a homologous, direct, competitive ELISA. The average numbers of high-affinity antibodies per fusion were 1.3 and 5.7 in the hyperimmunized and latent immunized groups, respectively. There was no significant difference in the specificities between the two immunization groups.

Importance of assisted reproductive technologies in the conservation of wild, rare or indigenous ungulates: review article.

Biodiversity is increasingly threatened by intensive agriculture, environmental pollution, extinction of natural habitats and several other factors. Several mammalian species including ungulates have disappeared or are threatened by extinction. However, ungulates play an important role both in the ecosystem and in the economy. In general, species or breeds are considered endangered if their population does not exceed 1,000 individuals. In these cases conservation programmes should be initiated in order to maintain or even increase their number. This review deals with the possibilities and limitations of assisted reproductive technologies (ART) in the conservation of ecologically valuable wild, rare and indigenous ungulates. The methods discussed here are artificial insemination, cryopreservation of semen and embryos, embryo recovery and transfer, in vitro production of embryos, as well as micromanipulation techniques including sperm injection, assisted hatching and cloning. Some of these procedures are already being exploited in the breeding of farm ungulates, but more basic information about the reproductive patterns of wild, rare and indigenous animal species is needed before the routine use of ARTs.

Studies on factors affecting superovulation and embryo transfer in Hungarian merino ewes.

The objectives of this study were (a) to assess the ovulatory response and embryo production of Hungarian Merino ewes after superovulation, (b) to investigate the factors influencing the efficiency of embryo transfer (ET) in Hungarian Merino ewes, (c) to compare the results of two ovarian stimulation protocols (PMSG and PMSG + FSH treatment) in Hungarian Merino ewes, and (d) to study how superovulation, laparoscopic insemination and surgical embryo retrieval (ER) affect the subsequent reproduction of Hungarian Merino donor females after an ET programme. There was no significant difference between the ovarian stimulation protocols in the ratio of donor ewes responding to superovulation nor in the average number of corpora lutea. However, the number of transferable embryos recovered per donor ewe was higher in the PMSG + FSH group. The proportion of transferable embryos, unfertilized oocytes and degenerated embryos did not differ between the treatment protocols. The total pregnancy rate was 53.4% (179/335). Neither the developmental stage of the embryo nor the number of transferred embryos affect the implantation of embryos. However, the increased number of transferred embryos positively influenced the pregnancy rate. No difference was found in the pregnancy rate between synchronised and non-synchronised groups of recipients. Thirty-six out of 45 donor ewes (80%) became pregnant within one year after the ET programme, indicating that ovarian stimulation and surgical ER did not affect adversely their reproduction.

Comparative study of different methods for dog semen cryopreservation and testing under clinical conditions.

The extenders and freezing rates from three different freezing protocols were combined and compared to each other in order to study the post-thawing acrosome integrity and fertility of frozen dog sperm. A commercial bovine TRIS-base extender (TRILADYL) and two self-made canine semen extenders (Norwegian and Dutch) were combined with a conventional bovine and two canine freezing regimes, and acrosome integrity of frozen/thawed spermatozoa was assessed by fluorescein isothiocyanate conjugated peanut agglutinin staining (FITC-PNA). Differences between freezing/thawing protocols were reflected in the proportion of cells with acrosomal damage and not based on motility results. It was concluded that during dog semen cryopreservation extenders had less influence on the post-thawing sperm quality than did the freezing rates. The optimal extender/freezing rate combination (TRILADYL/Norwegian) was used in the clinical practice to evaluate the fertility of frozen sperm administered by intrauterine insemination using a surgical approach. The pregnancy rate was 57% (4/7), but the average litter size was low (2.8). This may have been due to the insufficient sperm numbers contained in an insemination dose and/or to the incorrect timing of artificial insemination (AI). The final conclusion is that the commercial bovine extender is useful for freezing dog semen, and the TRILADYL/Norwegian freezing protocol is recommended as the most advantageous combination for the freezing of canine semen in the clinical practice.

Survival of mouse blastocysts after low-temperature preservation under high pressure.

Cryoinjuries are almost inevitable during the freezing of embryos. The present study examines the possibility of using high hydrostatic pressure to reduce substantially the freezing point of the embryo-holding solution, in order to preserve embryos at subzero temperatures, thus avoiding all the disadvantages of freezing. The pressure of 210 MPa lowers the phase transition temperature of water to -21 degrees C. According to the results of this study, embryos can survive in high hydrostatic pressure environment at room temperature; the time embryos spend under pressure without significant loss in their survival could be lengthened by gradual decompression. Pressurisation at 0 degrees C significantly reduced the survival capacity of the embryos; gradual decompression had no beneficial effect on survival at that stage. Based on the findings, the use of the phenomena is not applicable in this form, since pressure and low temperature together proved to be lethal to the embryos in these experiments. The application of hydrostatic pressure in embryo cryopreservation requires more detailed research, although the experience gained in this study can be applied usefully in different circumstances.

Assisted reproductive research: laser assisted hatching and spindle detection (spindle view technique).

Animal experiments are very important for the development of new assisted reproductive techniques (ART) for use in human and animal reproductive medicine. Most technical aspects of reproductive manipulation of humans and animals are very similar, and many components of successful human ART used nowadays have been derived from animal studies. In this study we examined (1) the use of 'non-contact' laser for assisted hatching, (2) whether spindles in living mouse oocytes could safely be imaged/examined by polarisation microscope (polscope) and (3) the influence of environment (e.g. temperature, in vitro culture, etc.) on spindle detection/visualisation. The data of the study presented here show that (1) laser assisted hatching (AH) is a fast, very accurate and safe procedure without any harmful effect on embryo development and it can support very effectively the implantation of embryos, (2) the use of polscope facilitates the evaluation of oocyte quality and the selection of oocytes with spindle, (3) by monitoring the spindle position during intracytoplasmic sperm injection (ICSI), we can reduce spindle damage and increase the chance of fertilisation. Further studies are underway to test the hypothesised connection between spindle birefringence and developmental capacity of oocytes/embryos.

Endoscopic methods in swine reproductive research: a review.

One of the major objectives of applying clinical methods is to get a more profound knowledge of the state of an organism. With the expansion of knowledge in physiology as well as the perfection of techniques the different applications have also improved. Over the past decade it has become obvious that in swine reproductive research such techniques are needed which do not influence the physiological processes and guarantee animal welfare. The minimal invasive and non-invasive methods (transabdominal endoscopy and ultrasonography) developed in human medical practice can be successfully adapted to the requirements of swine reproductive research. The present paper reports on the experience gained with abdominal endoscopy in swine so far.

Treatment of anoestrous mares with a synthetic progestagen, allyloestrenol.

Anoestrous mares were treated with prostaglandin (n = 43) and those that did not respond to prostaglandin (n = 29) with a synthetic progestagen, allyloestrenol, at a dose of 0.05 mg/kg body mass for 12 days. After the cessation of the long-term per os gestagen blockade the animals were checked for heat and, if a preovulatory follicle could be palpated, 2000 IU hCG was administered to induce ovulation. In some animals the plasma 17 beta-oestradiol (E2) and progesterone (P4) levels were also followed up throughout the gestagen treatment and for 10-14 days thereafter. As the favourable oestrus rate (86.2%) and pregnancy rate (65.5%) indicate, the 12-day allyloestrenol treatment seems to be a reliable tool for induction of cycling in mares anoestrous early in the season. These results, supported by the E2 and P4 profiles, suggest that an important reason for anoestrus in mares is the absence of a functional corpus luteum that can be substituted for by exogenous gestagen administration. The timing of ovulation, however, should still be improved.

Comparison of different methods used for oestrus examination in the bitch.

The aim of this study was to determine the reliability of oestrus symptoms of the bitch (vulvar swelling, vaginal fluid, attraction of the male, teasing) and that of the most frequently used examination methods (vaginal smear, vaginoscopy, serum progesterone determination) which indicate the stage of the cycle and allow to predict the optimal day of mating (ODM). The determination of the cycle stage was based upon the number of days left from the day of examination to the day of parturition (DTP). Our results indicate that none of the above-mentioned symptoms and examination methods is reliable alone for the determination of DTP, and even the complex picture obtained from the integrated results of different methods will not enable an exact estimation. ODM can be predicted with a higher reliability, and 1-2 days before it, it can be detected very safely. Since most bitches are presented for timing to practitioners, a scheme was created based upon a scoring system, which gives a point value to each individual symptom and examination result, and judges the bitch according to the total amount of points.

Induction of acrosome reaction in dog sperm by calcium ionophore.

The sensitivity of the plasma membrane to calcium ionophore (A23187) challenge was studied in dog sperm using fluorescein lectin staining for the assessment of acrosomal status and viability. Second fraction ejaculates from 5 dogs were washed, resuspended in Ca(2+)-free (EDTA-treated), 50, 100, 500, 1000 and 2000 microM/l Ca(2+)-containing Sp-TALP medium and induced with 50, 250, 500, 1000, 2500 and 5000 nM/l calcium ionophore. Samples were collected from each aliquot after 30 and 60 min of induction to assess the percentage of acrosome reacted sperm cells (AR rate), viability and motility by fluorescein isothiocyanate conjugated peanut agglutinin (FITC-PNA) and ethidium-homodimer combined staining. On each slide, 200 sperm cells were assessed under epifluorescence microscope (x 1250) in a blind manner. The response to ionophore challenge (AR rate, viability, motility) varied with Ca2+ and ionophore concentration in the suspension. A significantly higher AR rate was detected in samples containing 100, 500, 1000 and 2000 microM/L Ca2+ (> 40%) than in that containing 50 microM/L. Acrosome reaction could not be successfully induced in the EDTA-treated sample and in any of the aliquots in which 50, 250 and 500 nM/L ionophore concentrations were used for induction. Motility decreased drastically in all of the treated samples and stopped in that sample where as significant AR rate could be detected. Viability remained high (> 75%) during the incubation and did not differ significantly in the treated and the control groups.

Assessment of nutritional adequacy of the protein in dog foods by trials on growing rats.

In order to offer methods for assessing the protein quality of dog foods, eight commercial dry-type complete dog foods were analysed by in vitro chemical methods (crude protein, amino acid composition, in vitro pepsin digestibility) and rat growth trial (weight gain = WG, feed efficiency = FE, protein efficiency ratio = PER, net protein ratio = NPR, and net protein utilisation = NPU). Three individual samples were taken from each dog food and their homogenous mixture was used as an ingredient for the test diets fed to six rats per diet. All dog food samples were used as a single protein source in isonitrogenous (10% crude protein) and isoenergetic (ME = 14.9 MJ/kg) complete diets. Rat weight gains showed significant differences among most of the test diets as a consequence of the differences in protein quality of the dog food samples. The protein quality indices of the dog food products showed significant variations. The measured ranges of protein quality indices of the samples were as follows: WG = -5.4-21.4 g; FE = 0-0.25 g/g; PER = 0-2.54 g/g; NPR = 1.06-3.52 g/g; NPU = 19.93-65.93%. The limiting amino acid, the sum of essential amino acids and the chemical scores were calculated from gross amino acid composition. Tryptophan was the first limiting amino acids in all samples but one. Methionine was found to be the second limiting amino acid. The crude protein content of the samples showed no correlation with indices of protein quality, indicating that the declaration of crude protein is not a sufficient information to judge the protein quality of dog foods. The declaration of NPR, or NPU indices may be recommended for labelling the protein quality of premium-type dog foods.