L. Solti

Detection of calcium ionophore induced membrane changes in dog sperm as a simple method to predict the cryopreservability of dog semen

The sensitivity of dog sperm cells for extracellular Ca2+/Ca2+‐ionophore challenge was compared to the detrimental effects of an optimized freeze/thawing protocol. Three sperm‐rich fractions of ejaculates from 9 dogs were obtained, and one aliquot of each ejaculate was washed in a modified Tyrodes medium (HBT containing 0.1 mM Ca2+), without (control sample) and with 2.5 μM Ca2+‐ionophore (induced sample) and incubated for 60 min at 38°C in humidified atmosphere. Another aliquot from the same semen fractions was diluted, washed in a Tris buffer, and packed into 0.5‐ml straws with a Tris buffer containing 7.5 vol % glycerol. The samples were stored for 1 week in liquid nitrogen after a computer‐driven three‐step freeze protocol and subsequently thawed for 50 sec in a 37°C water bath and reconstituted into HBT. The acrosome integrity was determined using fluorescein‐conjugated peanut agglutinin (PNA‐FITC) as an acrosomal marker, while the vitality of the sperm cells was simultaneously assessed with the membrane impermeable DNA supravital stain ethidium homodimer 1 (EthD‐1) using fluorescence microscopy and flow cytometry. The motility of frozen/thawed sperm samples was evaluated by microscopic as well as computerized motility analyses. Remarkably, the percentage sperm cells that underwent acrosome reactions induced by Ca2+‐ionophore correlated very positively (r = 0.93) with the amount of acrosome damage observed in cryopreserved sperm samples. Furthermore, the degree of cellular damage induced by Ca2+‐ionophore treatment correlated very negatively (r = −0.99) with the relative amount of sperm cells that remained motile after cryopreservation. Such clear correlations between Ca2+‐ionophore induced acrosome reaction and motility parameters for frozen/thawed dog sperm cells were not found, suggesting that the generation of acrosome leakage and sperm immotility are two independent detrimental processes occurring during cryopreservation. From these results it can be concluded that Ca2+‐ionophore treatment followed by simultaneous determination PNA‐FITC and EthD‐1 staining can be used to predict the cryopreservability of ejaculates from individual dogs used as donors. Mol. Reprod. Dev. 55:289–298, 2000.

Tissue distribution of ochratoxin A as determined by HPLC and ELISA and histopathological effects in chickens

Ochratoxin A is a common feed contaminant, which may impair animal health and may lead to residues in edible tissues of slaughter animals. To simulate field conditions, broiler chicks were exposed to a total of 0.5 mg ochratoxin A per week for each of 4 weeks. Plasma toxin levels and tissue residues were measured by high-performance liquid chromatography (HPLC) and microplate enzyme-linked immunosorbent assay (ELISA). Results indicate an accumulation in plasma and wide distribution into all organs, with high levels in the liver and the kidney. Microscopical changes that could primarily be associated with toxin exposure were glomerulonephrosis, tubulonephrosis, focal tubular epithelial cell proliferation and multiple, adenoma-like structures in the renal parenchyma. The HPLC and ELISA methods gave similar results for both tissue distribution and depletion. Differences in absolute tissue toxin concentrations obtained by the two methods might be attributed to the different extraction and clean-up procedures, along with antibody specificity. The findings indicate that the dose applied causes subclinical tissue lesions and measurable tissue residues.

Evaluation of spermatological parameters in ochratoxin A—challenged boars

Exposure to certain mycotoxins has been proved to contribute to fertility problems in pigs. Although ochratoxin A (OA) is one of the most common naturally occurring mycotoxins, there is little data concerning the possible effects of this toxin on sperm quality of boars. After a 4-week control period, animals were given 20 microg OA per os daily for 6 weeks, followed by a 9-week withdrawal period. Serum and seminal plasma were monitored for OA with enzyme-linked immunosorbent assay (ELISA). Spermatozoal motility was measured at 0, 24, 48, 72 and 96 h, and ejaculation volume, initial viability and progressive motility were recorded. Samples of testis and epidydimidis were evaluated histologically. Viability, initial forward motility, and motility after 24h storage were significantly reduced in the experimental group in the withdrawal period only. There were no major histological differences in number and morphology of Leydig cells and epydidimal structures between experimental and control boars. Results of the present study demonstrate that OA may affect sperm production and boar semen quality only after a lag period. Further research is required to elucidate the possibility of a direct or indirect interaction between the toxin and germ cells (spermatogonia).

Artificial insemination of small ruminants — A review

Artificial insemination (AI) can undoubtedly be regarded as the oldest and most widely used assisted reproductive technique/technology (ART) applied in livestock production and it is one of the most important ARTs. The three cornerstones of its application are that it is simple, economical and successful. Artificial insemination offers many well-known benefits for producers. Fresh, fresh + diluted + chilled and frozen semen can be used for AI in small ruminants. To ensure its successful use, the AI technique must be selected on the basis of the type of semen planned to be used. This review paper gives a detailed overview of semen processing and its effects on semen quality, as well as of the AI techniques applied in small ruminants and their success rates.

Correlation between bull fertility and sperm cell velocity parameters generated by computer-assisted semen analysis

Motility is one of the most important characteristics associated with the fertilising ability of spermatozoa indicating their viability and structural integrity. Therefore, the examination of motility constitutes an integral part of semen analysis. Computer-assisted semen analysis (CASA) allows an accurate and objective assessment of different sperm motion characteristics with high repeatability. The aim of this study was to evaluate the different kinematic (velocity) parameters of frozen/thawed bull semen and determine if any of them could be correlated with their fertilising capability after insemination based on the achieved pregnancy rate. Ejaculates from 10 bulls were collected and frozen. The kinematic/velocity parameters of spermatozoa were measured by CASA and compared to the pregnancy results of almost 9,000 females artificially inseminated (AI) with frozen semen of any of the 10 tested bulls. The data of the experiments are summarised mainly with a focus on the effects of individual velocities (curvilinear velocity: VCL, straight-line velocity: VSL, average path velocity: VAP) on fertility rather than on the influence of progressive motility as a whole. We conclude that VAP is the most useful semen motility characteristic which has clinical relevance in the prediction of fertility.

Postpartal ovarian activity of healthy cows and those affected by subclinical metabolic disorders

Abstract The postpartal ovarian activity was studied in healthy cows and in those affected by subclinical fatty liver disease and subclinical primary ketosis. Twenty-six dairy cows, 4–7 years old, with not less than 5000 l milk yield in the preceding lactation, were monitored for the following blood parameters: aspartate-amino-transferase (AST)/glutamicoxalacetic transaminase (GOT), albumin, total protein, total lipid, cholesterol, non-esterified fatty acid (NEFA) and triglyceride levels, glucose and ketones. The ratios albumin : total protein and NEFA : triglyceride were also calculated. Blood samples were collected once a week between days 255 and 260 of gestation and days 1–15 postpartum, and subsequently on the 30th, 45th and 60th days postpartum. Based on the biochemical parameters, the cows were assigned in retrospect to three groups: (1) healthy ( n = 5), (2) subclinical fatty liver disease ( n = 6), and (3) subclinical primary ketosis ( n = 10). The postpartal ovarian activity was assessed by rectal palpation and by milk progesterone levels, determined by radioimmunoassay in samples collected 2–3 times weekly between days 5 and 70 postpartum. As early as 10–18 days postpartum, ovarian activity was evident in healthy cows. This was confirmed by the progesterone profiles indicating three regular ovarian cycles with normal hormone levels within 70 days postpartum. All five healthy cows were detected in estrus and only one failed to conceive after insemination. The six cows with subclinical fatty liver disease had no regular cycles and only a few small follicles were palpable on their ovaries. The milk progesterone fluctuated around the basal level (⩽ 1.6 nmol/l). The first Graafian follicles appeared as late as days 30–35 postpartum and the luteal function commenced only between days 38 and 60, as indicated by palpation of corpora lutea and a concomitant rise in milk progesterone. The progesterone peaks were at the lower physiological limits characteristic of luteal function. The ovarian activity of the ten cows with subclinical primary ketosis was intermediate between that of healthy and fatty liver cows. The first follicles and corpora lutea were somewhat delayed, and the progesterone levels fluctuated considerably during the luteal phase. One cow in this group resumed ovarian inactivity during the 70-day postpartal observation period.

Testicular function and semen characteristics of Awassi rams treated with melatonin out of the breeding season.

The objective of this study was to evaluate the effect of long-term melatonin treatment applied during the non-breeding season on semen characteristics, endocrine function of testicles and baseline level of insulin-like growth factor-I (IGF-I) in Awassi rams kept in the temperate continental zone of Europe and used as semen donors in an artificial insemination (AI) programme. On 23 February (day 0), slow-release melatonin implants were inserted subcutaneously into rams (n = 8). Control animals (n = 8) received no treatment. In both groups, basic semen parameters (concentration, total motility, fast and slow forward motility, morphology), GnRH-induced testosterone response and basal IGF-I concentration were evaluated on days 0, 47 and 71. No differences were found in concentration of spermatozoa, total motility, and numbers of spermatozoa with fast and slow progressive motility and normal/abnormal morphology between the melatonin-treated and the control group. However, in melatonin-treated animals, basal and GnRH-induced testosterone levels were slightly elevated on day 47 and became significantly higher on day 71 (P < 0.05) as compared to controls. There was no difference in plasma IGF-I levels between the groups. In conclusion, slow-release melatonin applied during the non-breeding season improves testicular testosterone production but does not influence the semen characteristics and the IGF-I level of semen donor Awassi rams used in an AI programme and kept in the temperate continental zone of Europe.

VITRIFICATION OF CLEAVAGE STAGE MOUSE EMBRYOS BY THE CRYOLOOP PROCEDURE

By decreasing the volume of the cryoprotective solution it is possible to increase dramatically the freezing speed and - at the same time - reduce the toxicity and osmotic side effects of cryoprotectants (CPA). The objective of our study was to vitrify Day-3 cleavage stage mouse embryos (n = 229) with the cryoloop technology using a new composition of vitrification media. Embryos were exposed to a 2-step loading of CPA, ethylene glycol (EG) and propylene glycol (PG), before being placed on the surface of a thin filmy layer formed from the vitrification solution in a small nylon loop, then they were rapidly submerged into liquid nitrogen. After warming, the CPA was diluted out from the embryos by a 3-step procedure. Survival of embryos was based on morphological appearance after thawing and continued development to expanded blastocysts upon subsequent 48-hour culture. Embryos of the two control groups were either treated likewise except that they were not vitrified, or cultured in vitro without any treatment. Our data show that a high percentage of embryos survived (92.7%) vitrification in the mixture of EG and PG combined with cryoloop carrier and developed normally (89.1%) in vitro after thawing. To our knowledge this is the first report of the successful vitrification of cleavage stage mouse embryos using VitroLoop vitrification procedure.

Survival of rapidly frozen hatched mouse blastocysts

The objective of the present study was to examine the effect of rapid freezing on the in vitro and in vivo survival of zona-pellucida-free hatched mouse blastocysts. Hatched blastocysts were rapidly frozen in a freezing medium containing either ethylene glycol (EG) or glycerol (G) in 1.5 M or 3 M concentration. Prior to freezing, embryos were equilibrated in the freezing medium for 2 min, 10 min, 20 min or 30 min at room temperature. To freeze them, embryos were held in liquid nitrogen vapour [approximately 1 cm above the surface of the liquid nitrogen (LN2)] for 2 minutes and then immersed into LN2. After thawing, embryos were transferred either to rehydration medium (DPBS + 10% foetal calf serum +0.5 M sucrose) for 10 minutes or rehydrated directly in DPBS supplemented with foetal calf serum. In vitro survival of embryos frozen with EG was higher than those frozen with G. The highest survival was obtained with 3 M EG and 2 min or 10 min equilibration prior to freezing, combined with direct rehydration after thawing. Frozen blastocysts developed into normal foetuses as well as unfrozen control ones did, with averages of 30% (control), 26% (EG) and 15% (G). The results show that hatching and hatched mouse blastocysts can be cryopreserved by a simple rapid freezing protocol in EG without significant loss of viability. Our data indicate that the mechanical protection of the zona pellucida is not needed during freezing in these stages.

In vitro survival of cryopreserved hatching or hatched in vitro produced (IVP) bovine embryos

On the international embryo market the presence of zona pellucida is required to decrease the risk of disease transmission. Following embryo manipulations, the embryos are no longer in an intact zona. In those cases the optimal stage and method for cryopreservation needs to be determined and might include the hatched blastocyst stage, too. The technical possibility for freezing in vivo hatched blastocysts have been demonstrated by the very fist results in bovine (Wilmut and Rowson, Vet Ret 1973;92:686). We compared a conventional freezing (Massip et al., Ann Med Vet 1987;131:51.5) and a vitrification method (Rall, Anim Reprod Sci 1992;28:237) on in vitro produced bovine embryos (Vajta et al.,Theriogenology 1992;37:811). Day 8, 9 and 10 (Fertilization= Day 0) hatching or hatched blastocysts were selected. In the freezing group, embryos were equilibrated for 10 min in 1.36 M glycerol and 0.25 M sucrose in PBS +lO% oestrous calf serum (OCS) at room temperature then loaded in 0.25 ml straws. Straws were sealed and placed at -7°C in the chamber of a freezing machine; after seeding and 10 min holding were cooled at a rate of 0.3’C/min to -25°C then plunged and stored in LN2. In the vitrification group embryos were equilibrated in VS3a (6.5 M glycerol and 6% BSA in PBl) in 3 steps at 20-250C (20 min in 25%, 1 min in 65% and then 1 min in a drop of 100% VS3a in the straw); cooled rapidly (2OO’C/min) and stored in LN2. In both groups straws were thawed in air then in 220C water for lo-10 set; cryoprotectant removal was performed in Petri dishes in 0.5 M sucrose for 5 ruin and rehydration was in PBS for 5 mitt at room temperature. Embryos were cultured further on granulosa cells in TCM 199 +10% OCS. Immediate survival was defined as the reexpansion of the blastocoel during rehydration. Twenty four h post-thaw the maintenance of blastocoel or the attachment to the monolayer with subsequent loss of blastocoel was examined. Rate of embryos growing further (>500ltm diameter of trophoblast layer and growing ICM) on the monolayer was recorded at 120 h post-thaw. Data were analyzed by x2 test. The immediate stuvival ranged between 95-100%. The 24 h results are presented in the table below. The rate of embryos growing further at 120 h ranged between lo-18%, treatment groups did not differed significantly.