Serological visualization of interleukin 2

Interleukin 2, a lymphokine that acts as a second signal of cellular immune response by way of its action as a T-cell growth factor, was morphologically identified by immunoperoxidase staining. With the use of a monoclonal antibody to interleukin 2 and several complex-forming antisera, the lymphokine was readily distinguished in cytocentrifuge preparations of peripheral blood leukocytes stimulated with a T-cell mitogen. When preparations of cloned interleukin 2 producer and responder cells were stained by the same procedures, discrete patterns of both responder and producer cell phenotypes were revealed. Interleukin 2 producer T cells exhibited a characteristic intense, ringlike cytoplasmic staining, whereas the responder cells (as exemplified by interleukin 2-dependent cell lines) exhibited a less intensive, spotlike membrane staining. In addition, intense membrane localization of interleukin 2, reminiscent of potential capping phenomena, could be observed in stained preparations of cloned responder cells.     

Effect of Estradiol and Progesterone on Metabolic Biomarkers in Healthy Bitches

The aim of this study was to evaluate the possible association between hormonal changes that occur during oestrus and biomarkers related with glucose metabolism (glucose and insulin), lipid metabolism (lipidic profile and BChE) and adipokines (adiponectin and ghrelin) in healthy bitches. For this purpose, we measured these analytes in serum of bitches, at two times: before (T1) and after (T2) the LH peak that were established according to progesterone concentrations. Increased levels of total cholesterol (p < 0.01), high density lipoprotein cholesterol (HDL‐C) (p < 0.01), low density lipoprotein cholesterol (LDL‐C) (p < 0.01), adiponectin (p < 0.01) and ghrelin (p < 0.05) were observed at T2 in comparison with T1. No statistically significant changes were observed in serum glucose, insulin, homoeostasis model assessment for insulin sensitivity (HOMA), triglycerides and BChE. When all data of T1 and T2 were pooled, serum adiponectin showed positive correlation with progesterone (r = 0.353; p = 0.022) and HDL‐C (r = 0.307; p = 0.048), and negative with insulin (r = ?0.429; p = 0.005), HOMA (r = ?0.446; p = 0.003) and BChE (r = ?0.522; p < 0.001). Ghrelin showed negative correlation with estradiol (r = ?0.701; p = 0.004). BChE was negatively correlated with estradiol (r = ?0.441; p = 0.018) and glucose (r = ?0.343; p = 0.028), and positively with insulin (r = 0.460; p = 0.003) and HOMA (r = 0.505; p < 0.001). In conclusion, changes in metabolic biomarkers occur in bitches after LH peak, characterized by increased lipids (total cholesterol, HDL cholesterol and LDL cholesterol) without changes in BChE activity, and increased adiponectin and ghrelin concentrations, without significant changes in glucose and insulin.     

Infertility in a Dog due to Proximal Cytoplasmic Droplets in the Ejaculate: Investigation of the Significance for Sperm Functionality In Vitro

A 4-year-old Basque Shepherd male dog was presented for breeding soundness evaluation after the dog failed to impregnate the three bitches he had mated. Clinical examination showed no anomaly of the reproductive system. Semen evaluation showed normal sperm count (640 x 10(6)), 80% had progressively motile spermatozoa, and 96% had morphologically abnormal sperm of which 84% had proximal cytoplasmic droplet and 12% had proximal droplet plus other anomaly. A zona pellucida-binding assay, using canine oocytes derived from frozen-thawed ovaries, was performed in order to investigate the zona-binding ability of dog spermatozoa with proximal cytoplasmic droplets. For the zona pellucida-binding assay, ovaries were thawed and minced in phosphate-buffered saline + 0.4% bovine serum albumin, the oocytes recovered were divided into two groups of 35-40 oocytes to be, respectively, used with the infertile dog and with a control fertile dog. Spermatozoa were capacitated in Canine Capacitating Medium (CCM) at 38.5 degrees C and 5% CO(2) in air for 2 h before oocyte insemination. Groups of five to six oocytes placed in 45 microl droplets of CCM were incubated for 1 h. Afterwards, 5 microl of CCM containing 25,000 spermatozoa were added to each droplet and co-incubated for 2 h before fixation and evaluation of the complexes. After oocyte insemination, sperm motility and viability were evaluated: the sample from the infertile dog had 85% sperm motility with fast and linear progressive movement, and sperm viability of 92%. The sample from the control dog showed 40% sperm motility with fast and highly curvilinear and erratic movement, high degree of sperm agglutination and sperm viability of 32%. For the infertile dog the mean number of bound spermatozoa/oocyte was 0.33 whereas for the control dog it was 1.80. It was concluded that dog sperm with proximal cytoplasmic droplets seem to lack normal capacitating ability in vitro, and consequently, they may have reduced capacity to bind to the zona pellucida of canine oocytes.     

Oestrous Synchronization,Ovarian Superovulation and Intraspecific Transfers From a Closed Breeding Colony of Inbred SLA Miniature Pigs

The inbred SLA miniature pig is a unique animal model developed for organ transplantation studies and pre‐clinical experimental purposes. Reported oestrous synchronization and superovulation treatments were examined in two SLA haplotypes (AA and DD) to allow collection of embryos for both practical embryo transfer and experimental technologies from a closed breeding colony. Pre‐puberal miniature pigs were poor responders to oestrous synchronization treatments, while post‐puberal sows were equivalent to commercial sows. Following superovulation, the ovulation number (corpora .hemorrhagica) was higher (p < 0.05) in the cycling sows when compared with non‐cycling sows. Ovulations were equivalent to commercial pre‐puberal gilts and non‐cycling sows (p > 0.05). No difference in ovulation number between haplotypes was observed, which differs from the previous report (DD>AA). Collection of zygotes for pronuclear injection was the highest in the non‐cycling post‐puberal miniature pig group (p < 0.05), although significantly lower when compared with the commercial pig treatment groups (p < 0.05). The incidence of cystic endometrial hyperplasia in our colony was equivalent to rates observed in commercial pigs. Pronuclear visualization following centrifugation was the highest in the non‐cycling miniature sow group and approximates to about 25% of ovulations and about half the rate observed in the commercial pigs (50%). Miniature pig embryos transferred between SLA haplotypes and transfer of DD embryos to commercial pigs resulted in live births at a higher efficiency than previously reported. This study demonstrates the feasibility of undertaking assisted reproductive technologies in a closed breeding colony of inbred SLA miniature pigs without compromise to the breeding programmes.     

A role for DNA characterisation in ovine footrot

Bacteroides nodosus involved in several outbreaks of ovine footrot over a number of years were subjected to DNA restriction endonuclease analysis. Individual isolates were found to have characteristic Bam HI profiles which permitted their accurate identification and differentiation from other isolates. Bam HI profiles of B. nodosus isolates were used in epidemiological investigations involving consecutive outbreaks of footrot on individual and neighbouring farms. The relationship of given isolates to a common source could be established by this means. Restriction endonuclease analysis provides an additional epidemiological tool in ovine footrot investigations as it accurately identifies interstrain differences in a manner not possible by conventional bacteriological and serological means.     

Oestrous sheep serum balances ROS levels to supply in vitro capacitation of ram spermatozoa

Reactive oxygen species (ROS) are fundamental for intracellular signalling. In spermatozoa, they are involved both to apoptosis and to capacitation, and changes in ROS levels can alter the balance between these two processes. Oestrous sheep serum (OSS) is considered an efficient agent for in vitro capacitation of ram spermatozoa. We have explored the effects of OSS on ram sperm physiology, especially on ROS production, during in vitro capacitation. Semen samples from 15 rams were cryopreserved. After thawing, samples were submitted to four treatments: control (CTL), 10% OSS supplementation for in vitro sperm capacitation, caspase inhibitor (INH, Z‐VAD‐FMK 100 μM) and OSS (10%) plus caspase inhibitor (I + E). Sperm samples were incubated for 30 min at 38.5°C and 5% CO₂ and evaluated motility and kinetic parameters by computer‐assisted semen analysis (CASA) and viability (propidium iodide), apoptotic‐like membrane changes (YO‐PRO‐1), acrosomal status (PNA‐FITC), intracellular calcium (FLUO‐3), membrane fluidity (M540) and ROS production (CM‐H₂DCFDA) by flow cytometry. OSS induced changes in kinetic parameters compatible with capacitation, with a decrease in the percentage of progressive motility and linearity, and an increase in the amplitude of the lateral displacement of the sperm head (p < .05). Moreover, OSS increased the proportion of M540+ viable spermatozoa, YO‐PRO‐1+ and acrosome‐reacted spermatozoa (p < .05). After incubation, OSS and I+E achieved lower ROS levels (p < .05). Ca²⁺ levels did not change with the incubation, but were slightly higher (p < .05) when both OSS and the inhibitor were present. We suggest that OSS may modulate ROS levels, allowing intracellular signalling for capacitation to occur while preventing higher levels that could trigger apoptosis.     

Influence of dietary lysine and energy intakes on body protein deposition and lysine utilization in the growing pig

A serial slaughter study was conducted to determine the effects of true ileally digestible lysine (IDLys) intake and metabolizable energy intake (MEI) on whole-body protein deposition (PD) and dietary lysine utilization in pigs between 45 and 75 kg live weight (LW). Conventional N balances were determined at the start and end of the serial slaughter study. Semisynthetic diets based on casein and cornstarch provided protein-bound lysine to support protein depositions of approximately 70% (Lys70%, IDLys 11.1 g/d) or 90% (Lys90%, IDLys 13.2 g/d) of a determined maximum PD. During the serial slaughter study and at Lys70%, pigs were fed one of six levels of MEI ranging from 14.1 to 23.5 MJ/d; at Lys90%, pigs were fed one of seven levels of MEI ranging from 15.6 to 26.4 MJ/d. The serial slaughter study and N balances indicated that MEI and IDLys had independent effects on PD and lysine utilization. Lysine utilization (calculated as the fraction of absorbed available lysine, over and above maintenance lysine requirements, that was retained in body protein) and PD increased with increasing MEI until plateau values were reached. At the plateaus, PD was determined by lysine intake. When lysine intake determined PD, lysine utilization did not decline (P > 0.10) with increasing lysine intake. Based on the N balance study, there was no effect (P > 0.1) of LW on lysine utilization. The marginal efficiency of using absorbed available lysine for PD was 0.75 and was not affected by LW, MEI, or IDLys.     

Influence of threonine intake on whole-body protein deposition and threonine utilization in growing pigs fed purified diets.

The relationship between available threonine (Thr) intake and whole-body protein deposition (PD) was established using the serial slaughter method in 36 individually housed growing gilts between 39 and 77 kg live BW. Pigs were prescreened for their maximum PD (PDmax), based on a N balance starting at 25 kg BW while they consumed semi-ad libitum a nonlimiting diet. Pigs were fed combinations of a casein and cornstarch-based diet that was confirmed to be first-limiting in Thr and a protein-free diet starting at approximately 30 kg BW. Casein-bound Thr was provided at 60, 70, 80, 90, 100, or 120% of estimated Thr requirements for PDmax. Energy intake was kept constant across treatments and exceeded requirements for PDmax. Pigs were fed three equal meals per day; feeding levels were adjusted weekly based on BW. Pigs were killed at either 39 kg BW (n = 2 per treatment) or 77 kg BW (n = 4 per treatment) for determining chemical body composition. Composition of 39-kg BW pigs was not different across treatments (P > 0.10); therefore, an overall mean initial body composition was used to estimate body protein content at the initial BW. Across treatments, mean daily ME intake was 25.3 (SE 0.08) MJ/d and did not differ (P > 0.10) among treatments. Average daily true ileal digestible Thr intake varied between 5.33 and 9.66 g/d, representing means for pigs on the lowest and the highest Thr intakes, respectively. Mean PD was 93, 102, 118, 124, 139, and 133 (SE 4.2) g/d for pigs on the six respective treatments. Dietary Thr intake did not influence (P > 0.10) Thr content of body protein at the final BW or the partitioning of body protein between carcass, viscera, and blood. The efficiency of Thr utilization for PD was lowest (P < 0.05) at the highest Thr intake level and highest (P < 0.05) at the lowest Thr intake level. It was similar (P > 0.10) at the four intermediate Thr intake levels, in which the relationship between true ileal digestible Thr intake and PD was linear. Based on these four treatments, calculated Thr disappearance, which is closely associated with inevitable Thr catabolism, was 23.5 (SE 0.55)% of available Thr intake. This value is consistent with an efficiency of using available Thr intake above maintenance Thr requirements (54 mg/kg BW0.75) for Thr retention with PD of 73.4 (SE 1.11)%. Based on N balances conducted at approximately 40 and 75 kg BW, the marginal efficiency of Thr utilization was not influenced by BW.     

Failure of beta-adrenergic receptor blockade to prevent arrhythmias induced by sympathetic nerve stimulation

Cardiac arrhythmias produced by electrical stimulation of the ventrolateral cardiac sympathetic nerve in dogs were not blocked by the combined administration of propranolol and practolol in amounts that completely blocked cardiac beta-adrenergic receptors. Blockade of cardiac alpha-adrenergic receptors, as well as cardiac cholinergic receptors, also had no influence on the arrhythmias. These results suggest that the adrenergic neuroeffector junction is fundamentally different from any hitherto described, differing perhaps in the neurotransmitter involved or in the nature of the receptor.