Recent advances in bovine sperm cryopreservation techniques with a focus on sperm post‐thaw quality optimization

Sperm cryopreservation facilitates the storage and transport of germplasm for its use in artificial insemination (AI) and other advanced reproductive technologies. The cryopreservation process can damage sperm and compromise functionality. Several cryobiological studies have found that the physical and biological factors that affect sperm survival at low temperatures during the cryopreservation process often involve the integrity of sperm membrane. In this review, the behaviour of the sperm membrane against cooling, cold shock, ice crystal formation, oxidative stress, osmotic changes, reorganization of the lipid bilayer and addition of cryoprotective agents (CPA) is discussed. In addition, the phenomenon of reactive oxygen species (ROS) and its relationship with the cryopreservation process is also described. Semen cryopreservation techniques have progressed slowly in past years, and the current performance, measured as post‐thawed survival, is not very different compared to past decades. Recent advances in understanding the structure of the cell membrane, its function and metabolism have driven to new conservation systems, including lyophilization and vitrification. However, none of these technologies is commercially available, although its future appears very promising.     

Investigation of the facilitation of the nociceptive withdrawal reflex evoked by repeated transcutaneous electrical stimulations as a measure of temporal summation in conscious horses

OBJECTIVE: To investigate whether facilitation of the nociceptive withdrawal reflex (NWR) can be evoked and quantified as a measure of temporal summation from the distal aspect of the left forelimb and hind limb in standing nonsedated horses via repeated stimulations of various subthreshold intensities and frequencies. ANIMALS: 10 adult horses. PROCEDURE: Surface electromyographic activity evoked by stimulation of the digital palmar and plantar nerves was recorded from the common digital extensor and cranial tibial muscles. For each horse, the NWR threshold intensity to a single stimulus was determined for the forelimb and hind limb. Repeated stimulations were performed at subthreshold intensities and at frequencies of 2, 5, and 10 Hz. The reflex amplitude was quantified, and the behavioral responses accompanying the stimulations were scored. RESULTS: Repeated stimulations at subthreshold intensities were able to summate and facilitate the NWR in conscious horses. The reflex facilitation was significantly related to the intensity of the repeated stimuli, whereas no effect of stimulation frequency was found. Reaction scores increased significantly for increasing stimulation intensities. CONCLUSIONS AND CLINICAL RELEVANCE: Temporal summation obtained by repeated stimulations of subthreshold intensity appears to represent a new tool for investigating nociceptive pathophysiologic processes in horses; this experimental model may be useful to examine the mode of action and efficacy of analgesic and anesthetic interventions and possibly to assess sensory dysfunction in clinical settings.     

Natural antimicrobials for control of Salmonella Enteritidis in feed and in vitro model of the chicken digestive process

This study evaluated the antimicrobial effect of essential oils (EO) and organic acids (OA) against Salmonella Enteritidis in chicken feed and during an in vitro model that mimics the chicken digestive process. The minimal inhibitory concentration (MIC) of allyl isothiocyanate (AITC), carvacrol (CV), propionic acid (PROP) and caproic acid (CAP) were individually determined. Then, based on the MICs of each compound, combinations of EOs and/or OAs were tested to evaluate their synergic antimicrobial effect. The synergic effect of AITC and CAP was the most efficient against the bacterial strain tested. Commercial feed was inoculated with a 5‐strain cocktail of S. Enteritidis and treated with different doses of AITC + CAP to evaluate their effect on the growth/survival of the pathogen. In addition, the simulated digestion model was used to access the antimicrobial effect of AITC + CAP added to the feed towards S. Enteritidis and Lactobacillus plantarum. Synergistic effect was found between AITC (0.065 mM) and CAP (17.5 mM) against S. Enteritidis in chicken feed, where S. Enteritidis was reduced to undetectable levels (<1.00 log CFU/g). AITC (1.95 mM) + CAP (45 mM) also decreased (p < 0.05) the population of S. Enteritidis in the simulated digestion, while the growth of L. plantarum was not affected. Therefore, the addition of AITC + CAP in feed might be a potential natural antimicrobial able to prevent economic losses caused for Salmonella in chicken.     

Vitamin E and polyunsaturated fatty acids in bovine muscle and the oxidative stability of beef from cattle receiving grass or concentrate-based rations

The present study was designed to assess the balance between antioxidant and prooxidant components and the oxidative stability of beef from cattle fed exclusively grazed pasture (PAS) or a barley-based concentrate offered indoors (CONC) for 11 mo, or fed grass silage indoors for a 5-mo winter period, followed for the remaining 6-mo summer period by grazed pasture (SiP) or by grazed pasture plus concentrate at 50% of the dietary DM (SiPC). Muscle prooxidant and antioxidant components were determined by measuring fatty acids and α-tocopherol concentration of LM, respectively. Lipid oxidation and color stability were monitored in ground LM, packaged in a high-oxygen modified atmosphere, over 11 d of refrigerated storage. Vitamin E concentration decreased (P < 0.0005) with an increasing proportion of concentrate in the diet (2.59, 2.45, 1.76, and 1.15 μg/g for PAS, SiP, SiPC, and CONC, respectively). A greater proportion of PUFA was found in LM from cattle in the PAS, SiP, and SiPC groups compared with animals in the CONC group (9.62, 11.04, 8.96, and 6.94%, respectively; P < 0.0005). A greater concentration of highly peroxidizable PUFA was found in LM from heifers in the PAS, SiP, and SiPC groups compared with those in the CONC group (0.84, 0.85, 0.87, and 0.65 mg/g of muscle, respectively; P = 0.02). Dietary treatment affected lipid oxidation (P < 0.0005), with greater 2-thiobarbituric acid reactive substance values in beef from heifers in the SiPC group than in beef from those in the PAS, SiP, and CONC groups. Dietary treatment affected myoglobin oxidation (P = 0.002) during storage, with greater metmyoglobin accumulation in beef from animals receiving concentrate (CONC and SiPC treatments) than in beef from cattle in the PAS and SiP groups. Consequently, feeding concentrate impaired meat color stability over the storage duration, with greater H* (hue angle) values (P < 0.0005) in meat from heifers in the SiPC and CONC groups compared with meat from those in the PAS and SiP groups. The results of the present study confirm a positive effect of grass-based feeding systems on meat color stability compared with concentrate-based dietary strategies. It appears that vitamin E in muscle alone does not explain the resistance of meat to oxidative deterioration because a clear interaction with highly peroxidizable PUFA exists.     

Performance and serum parameters of calves (Bos taurus) subject to milk restriction associated with supplementation with 2-hydroxy-4-(methylthio)butanoic acid

Our aim with this study was to evaluate the consumption, performance, quantitative characteristics of carcasses, biochemical profile, plasma levels of ghrelin and leptin, expression of the receptor for ghrelin (GHS-R1a) in the hypothalamus and duodenum, and the number of goblet cells in the duodenum of calves subjected to milk volume restriction and supplemented with 2-hydroxy-4-(methylthio)butanoic acid (HMTBa). We used 21 Holstein mixed-breed calves, aged between 3 and 15 d with an average weight of 36.8 kg, and housed in pens with troughs for hay, concentrate, and water. The study included two consecutive experimental periods (first period [P1] and second period [P2]) of 21 d each, with 7 d of adaptation to the diet and facilities. The calves were distributed in a completely randomized design in three treatments with seven repetitions. 1) Control: 6 liters of milk/d during P1 and 6 liters of milk/day during P2; 2) RES (milk restriction): 3 liters of milk/day during P1 and 6 liters of milk/day during P2; and 3) RES + HMTBa: 3 liters of milk/day during P1 and 6 liters of milk/day during P2 + 3.3 g of HMTBa/day in both periods. HMTBa was supplied in milk, and the amount of concentrated ration and hay provided and leftovers were recorded daily to estimate dry matter (DM) and crude protein consumption. Mean daily weight gain (DWG), final weight (FW), and feed conversion (FC) were obtained at the beginning and at the end of each 21-d period. Plasma concentrations of ghrelin and leptin, triglycerides, total protein, urea, lactate, creatinine, alkaline phosphatase, and cholesterol were measured for P1 and P2 at the end of each 21-d period. At the end of P2, animals were slaughtered; sections of the duodenum were collected to evaluate the expression of GHS-R1a and quantity of goblet cells; hypothalamus was used to evaluate the expression of GHS-R1a; rumen was used to evaluate the thickness of epithelium and keratin and the density, height, and width of ruminal papillae. In P1, total DM consumption, FW, DWG, glucose, and triglycerides were lower in the RES and RES + HMTBa groups (P < 0.001). In P2, there was an improvement in the FC of the RES + HMTBa group (compared with Control and RES groups) and a lower urea concentration in the RES group (compared with Control and RES + HMTBa groups) (P < 0.001). No differences were observed among groups regarding hormonal concentrations, histological parameters, and GHS-R1a expression in the duodenum and hypothalamus. Therefore, milk restriction combined with HMTBa supplementation promoted greater compensatory gain by a mechanism independent of changes in GHS-R1a expression and hormone levels of ghrelin and leptin.     

Evaluation of administration of isoflurane at approximately the minimum alveolar concentration on depression of a nociceptive withdrawal reflex evoked by transcutaneous electrical stimulation in ponies

OBJECTIVE: To investigate effects of isoflurane at approximately the minimum alveolar concentration (MAC) on the nociceptive withdrawal reflex (NWR) of the forelimb of ponies as a method for quantifying anesthetic potency. ANIMALS: 7 healthy adult Shetland ponies. PROCEDURE: Individual MAC (iMAC) for isoflurane was determined for each pony. Then, effects of isoflurane administered at 0.85, 0.95, and 1.05 iMAC on the NWR were assessed. At each concentration, the NWR threshold was defined electromyographically for the common digital extensor and deltoid muscles by stimulating the digital nerve; additional electrical stimulations (3, 5, 10, 20, 30, and 40 mA) were delivered, and the evoked activity was recorded and analyzed. After the end of anesthesia, the NWR threshold was assessed in standing ponies. RESULTS: Mean +/- SD MAC of isoflurane was 1.0 +/- 0.2%. The NWR thresholds for both muscles increased significantly in a concentration-dependent manner during anesthesia, whereas they decreased in awake ponies. Significantly higher thresholds were found for the deltoid muscle, compared with thresholds for the common digital extensor muscle, in anesthetized ponies. At each iMAC tested, amplitudes of the reflex responses from both muscles increased as stimulus intensities increased from 3 to 40 mA. A concentration-dependent depression of evoked reflexes with reduction in slopes of the stimulus-response functions was detected. CONCLUSIONS AND CLINICAL RELEVANCE: Anesthetic-induced changes in sensory-motor processing in ponies anesthetized with isoflurane at concentrations of approximately 1.0 MAC can be detected by assessment of NWR. This method will permit comparison of effects of inhaled anesthetics or anesthetic combinations on spinal processing in equids.     

Assessment of the Long-Term Viability of the Myxospores of Myxobolus cerebralis as Determined by Production of the Actinospores by Tubifex tubifex

While whirling disease was first observed in Rainbow Trout Oncorhynchus mykiss in 1893, the complete life cycle of Myxobolus cerebralis (Mc), the causative agent of the disease, was not understood until 1984, when it was shown to involve two obligate hosts, a salmonid fish and the aquatic oligochaete Tubifex tubifex (Tt). The viability of the triactinomyxon (TAM) actinospores produced by Tt has been well studied, and is known to be temperature dependent and measured in days and weeks. Assertions that Mc myxospores produced by infected fish remain viable for years or even decades were made during the mid-20th century, decades before the Mc life cycle was described. Moreover, the duration of myxospore viability has not been well studied since the life cycle was elucidated. In a series of time-delay treatments, we assessed the long-term viability of Mc myxospores by exposure to Mc-susceptible Tt oligochaetes and quantified TAM production. As the time delay between inoculation and incubation of Mc myxospores in sand and water and exposure to Tt oligochaetes increased, TAM production decreased exponentially. Production among the 15-d time-delay replicates was reduced 74.7% compared with the 0-d treatment. Likewise, total TAM production was reduced 94.5, 99.4, and 99.9%, respectively, in the 90-, 120-, and 180-d time-delay treatments. Linear regression analysis of our data and the absence of TAM production among replicates of Mc myxospores held at 5°C for 365 d prior to exposure to Mc-susceptible Tt oligochaetes indicate that the long-term viability of Mc myxospores is less than 1 year under the conditions of this study.

Received May 14, 2014; accepted September 27, 2014     

Performance,endocrine, metabolic,and reproductive responses of Nellore heifers submitted to different supplementation levels pre- and post-weaning

The present study was conducted to evaluate the effects of high and low supplementation levels pre- and post-weaning on performance, endocrine, metabolic, and reproductive responses of Nellore heifers. Fifty Nellore heifers with 132?±?9.9 kg average body weight (BW) and 138?±?19 days of age were supplemented from 4 to 14 months. The heifers were distributed into five supplementation plans: HH—6 g/kg of BW of supplement pre- and post-weaning, HL—6 g/kg of BW of supplement pre-weaning and 3 g/kg post-weaning, LH—3 g/kg of BW pre-weaning and 6 g/kg of BW post-weaning, LL—3 g/kg of BW pre- and post-weaning, and CC—control, no supplementation. Interactions were not significant (P?>?0.10). The level of supplement fed pre-weaning did not affect any of the performance variables evaluated at the end of the experiment (P?>?0.10). There was a significant effect of supplementation and level of supplementation fed post-weaning on average daily gain (ADG) and final BW (P?<?0.05). Overall ADG was also affected only by supplementation and level of supplement fed post-weaning (P?<?0.05) with animals receiving 6 g/kg of BW post-weaning gaining more weight. Follicular diameter was greater in animals that received 6 g/kg of BW post-weaning (P?<?0.05). In summary, performance, endocrine, metabolic, and reproductive variables evaluated in the current study were improved by the level of supplement fed post-weaning. Heifers receiving supplementation of 6 g/kg of BW post-weaning had greater responses, independent of the level received during the pre-weaning phase.