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

Effects of some respiratory and glycolytic inhibitors on mitochondrial functionality in bovine semen

Fertility potential of spermatozoa depends on maintenance of the mitochondrial membrane potential (ΔΨm) that provides energy for sperm hyperactivation immediately prior to successful fertilization. Mitochondrial structure and integrity are associated with sperm motility and reduced fertility, and measurement ofΔΨm may be suitable for determining bull semen quality. Mitochondrial membrane potential in four commercial AI bulls was assessed using JC-1 and propidium iodide in the presence of the glycolytic inhibitors 2-deoxy-D-glucose (DOG) and iodoacetamide (IAM) and the respiratory inhibitor valinomycin(VAL) to determine the maximumm ΔΨm at minimum incubation. Flow cytometry recorded ΔΨm within a unified population for all treatments that represented sperm with low and high ΔΨm respectively. Maximumm ΔΨ was seen at 40 min incubation. Mean high fluorescence intensity (MFI) (orange) was significantly greater for untreated compared to the treated sperm, at 40 and 80 min incubation, in both fresh and frozen hawed semen. In sperm treated with VAL and IAM, ΔΨm was lowered significantly, and the proportion of sperm with high: low ΔΨm ratio was higher in control and DOG- treated samples representing more active mitochondria. In samples treated with VAL and IAM, the ratio was reversed, representing loss in activity. Cryopreservation increased the high:low ΔΨm ratio only in bull 1 by 30% and lowered it in bulls 2, 3 and 4 by 20% - 70% compared to fresh semen. The rise in bull 1 may relate to be the product of sperm demonstrating a capacitation- like effect of the freeze-thaw process which stimulated sperm hyperactivity. We conclude that mitochondrial function was affected adversely on freeze-thaw process. The 40 min incubation is satisfactory for future studies. Furthermore, IAM, a known glycolytic inhibitor, has a similar effect to VAL, a recognized respiratory inhibitor, which should provide a basis for further research. © Shahani et al

Generation of islet-like cell aggregates from human non-pancreatic cancer cell lines

To explore a novel source for the derivation of islets, we examined the differentiation potential of human non-pancreatic cancer cell lines, HeLa (cervical carcinoma cell line) and MCF-7 (breast cancer cell line). These cells were subjected to a serum-free, three-step sequential differentiation protocol which gave two distinct cell populations: single cells and cellular aggregates. Subsequent analysis confirmed their identity as pancreatic acinar cells and islet-like cell aggregates (ICAs), as evidenced by amylase secretion and diphenylthiocarbazone staining respectively. Reverse transcriptase-PCR and immunocytochemistry assessment of the ICAs revealed the expression of pancreatic specific markers Ngn-3, Glut-2, Pax-6 and Isl-1. These ICAs secreted insulin in response to glucose challenge, confirming their functionality. We propose that ICAs generated from HeLa and MCF-7 cell lines could form a promising in vitro platform of human islet equivalents (hIEQs) for diabetes research