Data from 34 yearling Hereford or Angus bulls were used to investigate relationships of testicular size, quantitative rates of sperm production, Sertoli cell numbers, numbers of germ cells supported per Sertoli cell, and the efficiency of spermatogenesis to daily sperm output and seminal quality. Two ejaculates were collected by electroejaculation from each bull on each of 2 days/week throughout the study. The percentage of progressively motile sperm and the percentage of morphologically normal sperm were determined from aliquots of fresh semen. Additional aliquots of semen were frozen in glass ampules or plastic straws and subsequently evaluated for postthaw motility and percentage of sperm with intact acrosomes. Sertoli cell numbers, the numbers of germcells per Sertoli cell, and the efficiency of spermatogenesis were unrelated to the quality of fresh or frozen semen (P greater than 0.05). In first ejaculates, the numbers of sperm and motile sperm were related (P less than 0.05) to testicular parenchymal weight (r = 0.38, and 0.50), daily sperm production (r = 0.45 and 0.53), and spermatids per gram of testicular parenchyma (r = 0.35 and 0.34). Testicular parenchymal weight and daily sperm production also were related to daily sperm output and to the average daily motile sperm output of these bulls (P less than 0.05), but could account for less than 25% of the variability in these end points among bulls.

Although genetic manipulation of farm animals is of great interest for animal production and the pharmaceutical industry, its efficiency remains far from satisfactory. Pronuclear injection, which is the most widely used technique for such modification, mainly in mice, remains limited for this species. Some alternatives have been developed such as sperm mediated gene transfer, in which the spermatozoa are used as vectors for DNA delivery during in vitro fertilization. Mature sperm cells are able to spontaneously bind exogenous DNA molecules which may be internalized into sperm nuclei. Given the potential of sperm mediated gene transfer for livestock animals transgenesis, the aim of this study was to evaluate four methods of DNA uptake for sperm mediated gene transfer in bovine: incubation with DNA, plasma membrane alteration induced by calcium ionophore followed by incubation with DNA, electroporation and lipofection. Spermatozoa not exposed to exogenous DNA were used as control group. Cleavage, blastocyst and hatching rates were recorded at 72 hours post insemination (hpi), days 9 and 12 of embryo culture, respectively. Exogenous DNA-positive embryos were evaluated by PCR. No effect of treatment was observed on cleavage, blastocyst and hatching rates. In addition, percentage of DNA positive blastocysts did not differ among experimental groups. In spite of the low number of positive embryos, our results show that all treatments presented similar efficiencies for DNA delivery during in vitro fertilization. In conclusion, although the development rates were similar and constant in all groups, other factors such as exogenous DNA sequence, size and concentration should be considered to improve sperm mediated gene transfer.

The Dag defect is one of the primary morphological defects in sperm correlating with reduced fertility. This defect is found in the spermatozoa of many livestock species. The aim of the study was to assess the morphometry of the heads of normal sperm and sperm with the Dag defect in the semen of Duroc breeding boars. Sperm morphology was examined in ten ejaculates each from 12 Duroc boars. In total, 3,600 morphologically normal sperm and 838 sperm with the Dag defect were evaluated. The area, perimeter, length and width of the sperm head were measured and these basic morphometric parameters were used to calculate four additional shape indices characterising the sperm head, i.e. ellipticity, elongation, roughness and regularity. Sperm with this defect had markedly smaller heads, 0.32 μm shorter and 0.19 μm narrower than the heads of sperm with normal morphological structure. The heads of sperm with the Dag defect also had a 1.1μm smaller perimeter and a 2.5 μm² smaller surface area than the heads of morphologically normal sperm. The Dag defect is found in boar sperm irrespective of the age of the individual. It affects the morphology of the sperm head.

Introduction The addition of low-molecular-weight antioxidants during the freezing process improves post-thaw sperm quality. The high antioxidant potential of cryopreserved semen could have a positive effect on the motility, viability, and energy status of sperm cells and their ability to bind to the zona pellucida of oocytes. The aim of the study was to determine the effects of different concentrations and combinations of vitamins E and C in a semen extender on selected quality parameters of frozen-thawed canine spermatozoa. Material and Methods The experimental material was the semen of four mixed-breed dogs. Sperm viability (motility, plasma membrane integrity, and mitochondrial function) was examined at 0, 60, and 120 min in semen samples supplemented with the extender and in the controls. Results Combined supplementation with vitamins C + E at a concentration of 200 + 200 μM /1 × 10⁹ spermatozoa had the most profound effect on total sperm motility, linear motility, and the percentage of spermatozoa with intact plasma membrane and active mitochondria. Conclusion The synergistic activity of vitamins E and C had a more beneficial influence on the quality of frozen–thawed sperm than these non-enzymatic antioxidants applied separately.

Introduction: The effect of two smear staining methods on the dimensions and shape of sperm cells in the semen of domestic pigs was evaluated. Material and Methods: The studies were carried out on 30 ejaculates collected from 15 boars, which included five Duroc boars, five Pietrain boars, and five hybrid Duroc × Pietrain boars. Each ejaculate was next sampled to make two microscopic slides, of which one was stained with eosin-nigrosin and the other with eosin-gentian dye. In total, 600 measurements of sperm cells were made. Each sperm was measured for the following morphometric parameters: head length, head width, head area, head perimeter, tail length, and the total sperm length. Results: Sperms measured on slides stained with eosin-nigrosin showed lower dimensions as compared with those stained with the eosin-gentian dye method. Sperm stained with eosin-nigrosin had shorter and narrower heads than sperm stained with eosin-gentian dye. The method of staining, therefore, affected not only the dimensions of the sperm, but also the proportions of the dimensions defining the shape of the sperm. Conclusions: The size and shape parameters in porcine sperm may take on different values depending on the method of semen staining. Sperm cells stained with eosin-nigrosin are smaller than the sperm stained with eosin-gentian dye. The sensitivity of the sperm to the type of dye used for the fixation may be associated with genetic factors.

Introduction: Significant improvement of sperm motility within one month effected by oral supplementation of selenium and vitamin E was described in four infertile male dogs which failed to conceive in their last three matings with different bitches. Material and Methods: The dogs (a Golden Retriever, an English Cocker Spaniel, and two Tibetan Mastiffs) were supplemented daily with selenium (Se) (0.6 mg/kg organic Se yeast) and vitamin E (vit. E) (5 mg/kg) per os for 60 days. Semen was collected on days 0, 30, 60, and 90. The sperm concentration and motility parameters were evaluated by the CASA system, sperm morphology was explored by Diff-Quick staining, and live and dead spermatozoa were differentiated by eosin/nigrosin staining. The concentrations of Se and vit. E were measured in peripheral blood serum on semen collection days. Results: Before administration, the concentrations of Se in blood plasma were low (86.0–165.0 µg/L). After 30 days of treatment there was an observable improvement in total and progressive sperm motility and kinematic parameters (VAP, VSK, VCL, ALH, BCF, and RAPID). The percentages of live and normal morphology sperm cells were also higher. There was also an observable increase in Se and vitamin E concentrations in blood serum. Bitches were successfully mated and delivered four to six puppies. Conclusion: Supplementation with Se and vit. E improved rapid sperm motility and restored fertility in infertile dogs with low Se status.

Introduction: The aim of this study was to evaluate the dependence between ejaculate traits, sperm morphology, and ejaculate volume in Duroc boars. Material and Methods: The analysis involved 121 ejaculates collected from 12 Duroc boars kept in three artificial insemination centres located in central Poland. Ejaculates were collected manually at one-month intervals, over a period of 10 months. At least 10 ejaculates were collected from each boar. The material was divided by ejaculate volume and each ejaculate was assigned to one of three volume groups: 160 mL and lower, 161–200 mL, and 201 mL and higher. The ejaculates were assessed to identify the basic physical traits and determine the incidence of morphological abnormalities in the spermatozoa, specifying major and minor abnormalities. Furthermore, the morphological structure indices for the spermatozoa were also calculated. Results: In large-volume ejaculates, spermatozoa were more elongated in shape, their heads were more elongated and had the largest flagella. With an increase in the ejaculate volume, sperm concentration in the ejaculate decreased. Moreover, while the total number of sperm in the ejaculate increased, the number of insemination doses obtained from a single ejaculate were higher. Conclusion: The volume of ejaculate has little impact on the occurrence of morphological abnormalities and the size of sperm cells. Ejaculate volume is important for the shape of the sperm cells.

Introduction: The aim of this study was to evaluate the effect of lipoprotein fraction isolated from ostrich egg yolk (LPFo) on the metabolic activity of boar spermatozoa following liquid semen storage in different extenders and temperatures. Material and Methods: Boar ejaculates were extended in Androhep, Beltsville thawing solution (BTS), and Martín-Rillo and Alias (MR-A) without (control) or with the addition of LPFo and stored for three days at either 5°C or 16°C. The analysed sperm parameters included total motility (TMOT), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), oxygen consumption, and adenosine triphosphate (ATP) production. Results: The sperm metabolic activity seemed to be higher in the LPFo-based extenders following storage for three days, irrespective of the storage temperature. Compared with the LPFo-free extenders, significantly higher (P < 0.05) sperm PMI and MMP were observed in BTS and MR-A extenders supplemented with LPFo during storage for three days at 5°C. Spermatozoa stored in the BTS-LPFo extender exhibited higher (P < 0.05) TMOT and oxygen consumption, whereas higher (P < 0.05) PMI was observed in spermatozoa stored in Androhep-LPFo and MR-A-LPFo for three days at 16°C. No significant differences (P > 0.05) in ATP content were observed between the LPFo-free and LPFo-based extenders during storage. Conclusions: Supplementation of LPFo to semen extenders had varying effects on the metabolic activity of boar spermatozoa stored at different temperatures. It can be suggested that the interactions of various components of the extenders and seminal plasma with LPFo exert beneficial effects on the sperm metabolic activity during liquid storage of boar semen.

Introduction: The aim of this study was to propose the optimal methodology for stallion semen morphology analysis while taking into consideration the staining method, the microscopic techniques, and the workload generated by a number of samples. Material and Methods: Ejaculates from eight pure-bred Arabian horses were tested microscopically for the incidence of morphological defects in the spermatozoa. Two different staining methods (eosin-nigrosin and eosin-gentian dye), two different techniques of microscopic analysis (1000× and 400× magnifications), and two sample sizes (200 and 500 spermatozoa) were used. Results: Well-formed spermatozoa and those with major and minor defects according to Blom’s classification were identified. The applied staining methods gave similar results and could be used in stallion sperm morphology analysis. However, the eosin-nigrosin method was more recommendable, because it allowed to limit the number of visible artefacts without hindering the identification of protoplasm drops and enables the differentiation of living and dead spermatozoa. Conclusion: The applied microscopic techniques proved to be equally efficacious. Therefore, it is practically possible to opt for the simpler and faster 400x technique of analysing sperm morphology to examine stallion semen. We also found that the number of spermatozoa clearly affects the results of sperm morphology evaluation. Reducing the number of spermatozoa from 500 to 200 causes a decrease in the percentage of spermatozoa identified as normal and an increase in the percentage of spermatozoa determined as morphologically defective.