Clinical, ultrasonographic and pathological findings in a bull with segmental aplasia of the mesonephric duct.

On assessment for use in an AI stud, a 12-month-old bull was found to produce low volume ejaculates with 41% of the sperm having morphological abnormalities. No left epididymal tail was palpable and the head of the epididymis on the left was twice the size compared with the right. Ultrasound examination showed the left testis to contain a large central area of decreased echogenicity, which could be followed proximally to a 15-mm echolucent lesion at the site of the epididymal head. Postmortem examination revealed a 15-mm diameter cyst in the region of the left epididymal head, and absence of the body and tail of the epididymis. The mediastinum testis of the left testis was dilated, corresponding to the area of decreased echogenicity observed on ultrasonography. No left seminal vesicle was present and the ampulla was significantly smaller than the same structure on the right. Histological examination revealed incomplete or absent spermatogenesis involving the majority of seminiferous tubules in the left testis, and a small proportion of those of the right testis. The cystic structure at the site of the left epididymal head was lined by irregular, sometimes attenuated, epithelium and contained sparse spermatozoa. This case demonstrates the adverse impact, which segmental aplasia of the mesonephric duct had on the testicular and epididymal function of a bull, and highlights the importance of careful clinical assessment in its diagnosis.

Dilution of spermatozoa results in improved viability following a 24 h storage period but decreased acrosome integrity following cryopreservation.

This study was designed to investigate the physiological factors affecting the reduced viability of cryopreserved spermatozoa following dilution. Ninety-six ejaculates were collected from 13 bulls and diluted to 10 x 10(6) and 60 x 10(6) sperm/ml in a commercial long term extender (Eqcellsire; IMV) and in an egg yolk extender. Samples diluted in the egg yolk extender were frozen in 0.25 ml straws. Samples diluted in the Eqcellsire were stored at room temperature for 24 h and assessed for sperm cell viability using SYBR14 and PI (Molecular Probes) and osmotic resistance. Frozen samples were thawed and assessed for viability, osmotic resistance and acrosome intergrity. Acrosome integrity was measured using Mitotracker, PI and PNA-FITC (Molecular Probes). Spermatozoa diluted to 10 x 10(6) sperm/ml and stored at ambient temperature had a higher proportion of viable spermatozoa (P < 0.01) and were less susceptible to osmotic stress (P < 0.01) than sperm diluted to 60 x 10(6) sperm/ml. Following cryopreservation there was no concentration-related difference in the proportion of viable spermatozoa and their relative susceptibility to osmotic stress. Spermatozoa diluted to lower cell concentrations had a higher proportion of viable cells that were acrosome reacted (P < 0.001). It is suggested that the higher proportion of acrosome reacted cells may result from an increased proportion of cells in a capacitated-like state in the spermatozoa diluted to lower concentrations. A Spearmans ranked correlation demonstrates a relationship between individual bull spermatozoa following dilution or cryopreservation for viability (r2 = 0.98; P < 0.001) or osmotic resistance (r2 = 0.87; P < 0.001) suggesting a variation in these characteristics between bulls.

Impact of antifreeze proteins and antifreeze glycoproteins on bovine sperm during freeze-thaw.

There are no reports on the use of antifreeze proteins (AFP) and antifreeze glycoproteins (AFGP) for the use of bull sperm cryopreservation despite studies in the ram, mouse and chimpanzee. The effect of freezing and thawing on bull sperm viability, osmotic resistance and acrosome integrity were observed following the addition of AFP1, AFPIII and AFGP at four concentrations (0.1, 1, 10 and 100 microg/ml). In a second part of the experiment, fluorescein was conjugated to the AFPs and AFGP and observations were made using fluorescence microscopy to determine whether binding occurred between the sperm cell membranes and the proteins. In the final part of the study the cryopreservation media were cooled in the presence of the AFPs and AFGPs at the four concentrations on a cryomicroscope to mimic similar cooling curves as those used in the presence of sperm. Following freeze-thaw, AFPI resulted in increased osmotic resistant cells at 0.1-10 microg/ml compared to the control (P<0.01). AFPI and AFPIII did bind to the sperm cells. There was no visual difference in ice structure between the control, AFPIII and AFGP but AFPI resulted in parallel crystals at 0.1, 1 and 10 microg/ml. We suggest that the increased osmotic resistance in the spermatozoa cryopreserved in AFPI is due to the cells orientating between the ice crystals, reducing mechanical stress to the cell membrane. Previous research has shown that osmotic resistance correlates with bull fertility, suggesting that bull spermatozoa cryopreserved in the presence of AFPI may have increased fertility in vivo.

Morphological defects of the acrosome in boar spermatozoa.

Morphological examination of semen from 17 boars of five breeds showed the presence of acrosome defects in 11 boars from four breeds. Two distinct types were seen; 'knobbed' sperm (type 1), of which two forms were found to be present by electron microscopy, and an uneven swellling of part of the acrosome (type 2) whose contents consisted of cytoplasmic and membrane-like material. The incidence of 'knobbed' sperm ranged from 0.2 to 6.3 per cent. Type 2 abnormalities were seen in only two boars, at 0.66 and 1.33 per cent.

Some observations on the semen of bulls persistently infected with bovine virus diarrhoea virus.

As a result of screening procedures employed for animals entering the AI service, two bulls were identified as being persistently infected with bovine virus diarrhoea virus by isolation of the virus from blood. Semen was collected on two occasions from these bulls; its quality as measured by density and motility was poor. Gross abnormalities of the sperm head, termed 'collapsed' heads, were seen in 28 to 45 per cent of sperm from one bull and in 1 per cent of sperm from the other. The collapsed heads were small and the whole head or its anterior part had the appearance of a dried pea. Electron microscopy showed the defect to consist of convoluted nuclear material with membrane-bound vacuoles and invaginations containing membranous debris and lamellar structures. In the 'high incidence' bull there was a corresponding increase in enlarged sperm heads. The 'low incidence' bull had sperm with heads of similar mean size to sperm from control bulls but with an increased variance. The semen was diluted in a lactose diluent, frozen and stored in liquid nitrogen. The distribution of viral antigen was determined and virus was isolated from several fractions of the semen, both before and after processing and cryopreservation. In one animal raw semen failed to yield virus but virus was recovered after processing, suggesting that raw semen may not be suitable for the efficient detection of the virus.

Mid-piece length of spermatozoa in different cattle breeds and its relationship to fertility.

Recently positive correlation has been found between oxygen consumption (ZO2) in bull spermatozoa and non-return rates and concluded that an increase in ZO2, characteristic of the freeze/thaw process, was possibly associated with mitochondrial membrane damage during this procedure: alternatively, sperm may be hyperactivated through the capacitation-like effects of freezing/thawing. We speculated that the morphology of spermatozoa may be associated with their rate of ZO2 and fertility: for example, sperm mid-piece length where mitochondria are located. Such a relationship has not been investigated before, particularly in context of commercial cattle breeding programmes and bull fertility characteristics. Sperm biometry was performed on ejaculates obtained from 34 bulls representing six breeds: Holstein (yearlings and mature), Friesian, Belgian Blue, Aberdeen Angus, Charolais and Limousin. Five ejaculates were collected from every bull and from each sample a semen smear was fixed and stained with eosin/nigrosin: the mid-piece length of 40 sperm with normal morphology was measured in every sample. Data were analysed by breed, age and within each bull. Significant differences (p<0.01) between ejaculates in 9/34 bulls was found, as well as differences (p<0.001) between individual bulls within the same breed. The average mid-piece length for Aberdeen Angus was 13.35 microm, for Belgian Blues and Limousin around 13.8 microm and for Charolais 13.68 microm: for dairy breeds such as Holstein and Friesian it was about 13.4 microm. The mean value of mid-piece length for breed was compared with their 49 day non-return rate; a negative correlation (r = -0.53) was found in black and white dairy breeds.

A photographic method for the measurement of motility of bull spermatozoa.

Bull spermatozoa were diluted in skim milk-egg yolk and frozen. After thawing, the samples were added to citrate buffer and photographed (1 sec exposure, 400 ASA, dark field) to identify the tracks of the moving spermatozoa. The proportions of motile spermatozoa in 1707 photographs of semen samples from 25 ejaculates were distributed binomially, and allowed motility to be estimated at a predictable level of precision, and without bias when one photograph from each of two straws was taken at random from an ejaculate. The variance was equal to its expectation and inversely proportional to the total number of spermatozoa in each photographic field.