Preliminary Evaluation of a Unique Freezing Technology for Bovine Spermatozoa Cryopreservation

Artificial insemination (AI) and semen cryopreservation has significantly improved the breeding potential of male animals. However, current freezing techniques commonly result in reduced semen quality. Ten years ago, a unique freezing technology (UFT) was developed for the freezing of foodstuffs and other materials. Previous work from this laboratory has demonstrated the UFT to be a superior method of freezing for a number of cell types. In a preliminary study, the UFT was compared with the conventional freezing methodology of bovine semen. Semen samples were collected from an angus (Bull A) and a gelbivich bull (Bull B), prepared using a conventional bovine cryoprotectant, and frozen in the UFT or in liquid nitrogen (LN) mist. The samples were stored in LN before being thawed and assessed for the semen parameters of motility and forward progression. Preliminary results suggest the UFT is equivalent to current techniques in the cryopreservation and recovery of bovine semen, and with modification, possibly a superior technique for semen freezing. Further studies using larger sample populations, and using a CASA system to evaluate motility, forward progression and linearity are merited.     

Effect of pyruvate and lactate on motility of cold stored stallion spermatozoa challenged by hydrogen peroxide

Numerous workers have shown that motility of cold stored spermatozoa from some stallions can be improved by removing most of the seminal plasma by centrifugation and resuspending the spermatozoa in an extender consisting of skim milk glucose extender (SKMG) supplemented with a salt media such as Tyrode's or phosphate-buffered saline. The salt media must contain pyruvate and lactate. In an effort to test the hypothesis that pyruvate may be acting as an antioxidant, a series of experiments were conducted using a H₂O₂ challenge to artificially produce damage due to lipid peroxidation. Results of these experiments indicated that addition of lactate or pyruvate and lactate to SKMG-Tyrode's media was not able to prevent the detrimental affects of H₂O₂. The addition of lactate to the SKMG-Tyrode's media resulted in an improvement of post-storage motility; however, increasing the concentration of pyruvate did not further improve motility. Therefore, because lactate dehydrogenase has been shown to be correlated with motility, and lactate has been shown to be preferred as an energy source by spermatozoa from other species, the beneficial effects of lactate and pyruvate as components of a modified SKMG extender are probable as energy sources.     

Board-invited review: Peptide absorption and utilization: Implications for animal nutrition and health

Over the last 50 yr, the study of intestinal peptide transport has rapidly evolved into a field with exciting nutritional and biomedical applications. In this review, we describe from a historical and current perspective intestinal peptide transport, the importance of peptides to whole-body nutrition, and the cloning and characterization of the intestinal peptide transporter, PepT1. We focus on the nutritional significance of peptide transport and relate these findings to livestock and poultry. Amino acids are transported into the enterocyte as free AA by a variety of AA transporters that vary in substrate specificity or as di- and tripeptides by the peptide transporter, PepT1. Expression of PepT1 is largely restricted to the small intestine in most species; however, in ruminants, peptide transport and activity is observed in the rumen and omasum. The extent to which peptides are absorbed and utilized is still unclear. In ruminants, peptides make a contribution to the portal-drained visceral flux of total AA and are detected in circulating plasma. Peptides can be utilized by the mammary gland for milk protein synthesis and by a variety of other tissues. We discuss the factors known to regulate expression of PepT1 including development, diet, hormones, diurnal rhythm, and disease. Expression of PepT1 is detected during embryological stages in both birds and mammals and increases with age, a strategic event that allows for the immediate uptake of nutrients after hatch or birth. Both increasing levels of protein in the diet and dietary protein deficiencies are found to upregulate the peptide transporter. We also include in this review a discussion of the use of dietary peptides and potential alternate routes of nutrient delivery to the cell. Our goal is to impart to the reader the nutritional implications of peptide transport and dietary peptides and share discoveries that shed light on various biological processes, including rapid establishment of intestinal function in early neonates and maintenance of intestinal function during fasting, starvation, and disease states.     

Uptake of zinc from zinc sulfate and zinc proteinate by ovine ruminal and omasal epithelia

Uptake and transport of Zn from (65)Zn-labeled ZnSO(4) and Zn proteinate (ZnProt) by ruminal and omasal epithelia were examined by using a parabiotic chamber system. Uptake was measured during a 4-h incubation with 10, 20, or 200 microM Zn as ZnSO(4) or ZnProt in the mucosal buffer (pH 6.0, Krebs-Ringer phosphate). Zinc uptake and transport were also evaluated after simulated ruminal digestion. Buffered ruminal fluid contained a feed substrate and 10 or 200 microM added Zn as ZnSO(4) or ZnProt. In a preliminary experiment, uptake of Zn by omasal tissue was low; thus, the remaining experiments were conducted solely with ruminal epithelium. Incubations to determine the effect of time on Zn uptake from mucosal buffer containing 20 microM added Zn as ZnSO(4) or ZnProt resulted in increased (P < 0.01) Zn uptake as incubation time increased from 30 to 240 min. Zinc uptake was also greater (P = 0.02) from mucosal buffer containing ZnProt compared with ZnSO(4). Zinc uptake from incubations containing 10 or 200 microM was affected by source x concentration (P = 0.05) and concentration x time (P < 0.01) interactions. With 10 microM Zn, uptake was not influenced by Zn source, whereas when 200 microM Zn was added, Zn uptake from ZnProt was greater than from ZnSO(4). Increasing incubation time resulted in increased Zn uptake with 200 microM Zn in the mucosal buffer; however, with 10 microM Zn, uptake did not change after 30 min. After simulated ruminal fermentation, the proportion of Zn in a soluble form was influenced by a source x concentration interaction (P = 0.03). After 18 h of incubation, the proportion of Zn that was soluble was not different between ZnProt and ZnSO(4) in buffered ruminal fluid that contained 10 microM added Zn, but was greater for ZnProt compared with ZnSO(4) with 200 microM Zn in the incubation. Zinc uptake from the aqueous fractions of simulated ruminal digestions containing 200 microM added Zn was greater (P < 0.01) than from those containing 10 microM added Zn. Zinc transport, based on detection of (65)Zn in serosal buffer, did not occur in any of the experiments. The results of the current experiments suggest that absorption of Zn into the bloodstream does not occur from the ruminant foresto-mach; however, Zn uptake occurs in ruminal tissue and is greater from ZnProt than from ZnSO(4).     

Validation of a Field Based Chromatin Dispersion Assay to Assess Sperm DNA Fragmentation in the Bottlenose Dolphin (Tursiops truncatus)

Over the last two decades, there have been significant advances in the use of assisted reproductive technology for genetic and reproductive management of captive dolphin populations, including evaluation of sperm DNA quality. This study validated a customized sperm chromatin dispersion test (SCDt) for the bottlenose dolphin (Tursiops truncatus) as a means of assessing sperm DNA damage both in the field and in the laboratory. After performing the SCDt, two different sperm morphotypes were identified: (i) sperm with fragmented DNA showed large haloes of dispersed DNA fragments emerging from a compact sperm nucleoid core and (ii) sperm containing non‐fragmented DNA displayed small compact haloes surrounded by a dense core of non‐dispersed DNA and protein complex. Estimates of sperm DNA fragmentation by means of SCDt were directly comparable to results obtained following a two‐tailed comet assay and showed a significant degree of correlation (r = 0.961; p < 0.001). This investigation also revealed that the SCDt, with minor modifications to the standard protocol, can be successfully conducted in the field using a LED florescence microscopy obtaining a high correlation (r = 0.993; p = 0.01) between the data obtained in the laboratory and in the field.     

Low-temperature tolerance of Nile tilapia, Oreochromis niloticus: effects of environmental and dietary factors

This study was conducted (1) to evaluate the effects of photoperiod (fixed vs. decreasing light), fish size (136 vs. 220 mm), dissolved ions (hardness and salinity) and diet (menhaden oil vs. coconut oil‐based) on the tolerance (survival) of Nile tilapia, Oreochromis niloticus, to low temperatures (decreased by approximately 0.5 °C per day) and (2) to evaluate the effect of dietary fatty acid composition on selected physiological characteristics of Nile tilapia exposed to decreasing temperatures. Size significantly affected mortality, with smaller fish being less tolerant to low temperatures than larger fish. Results were equivocal in the photoperiod, dissolved ion and dietary lipid experiments, and were dependent on the method of data analysis employed. Diet significantly affected plasma osmolarity, with higher values in fish fed the menhaden‐based diet. Haematocrit, serum glucose, sodium and cortisol concentrations, serum and splenic lysozyme activities, lymphocyte count and hepatosomatic index were not affected by diet. Haematocrit, white cell count and serum glucose and sodium concentrations were significantly affected by temperature, but serum and splenic lysozyme content, hepatosomatic index, and serum cortisol concentrations were not. The results of this series of experiments indicate that altering the environment or diet has little effect on the ability of Nile tilapia to survive low temperatures.