Ejaculate Fractioning Effect on Llama Sperm Head Morphometry as Assessed by the ISAS® CASA system

South American camelid sperm characteristics are poorly known compared with those of other domestic animals. The long‐term duration of ejaculation makes difficult to gather all the seminal fluid, implying possible ejaculation portion losses. Thus, the aim of this research was to evaluate the characteristics of the morphology and morphometry of the spermatozoa change during ejaculation. The morphometric characterization was tested on nine specimens of the Lanuda breed, using a special artificial vagina. In five of the animals, a fractioning of the ejaculate was performed by taking samples every 5 min. for a total of 20 min. Air‐dried seminal smears were stained with Hemacolor and mounted permanently with Eukitt. Morphometric analysis was carried out with the morphometry module of the ISAS^® CASA system. Almost 350 cells were analysed per sample, with a total number of 3207 spermatozoa. Mean values were given as follows: length: 5.51 μm; width: 3.38 μm; area: 17.75 μm²; perimeter: 14.8 μm; ellipticity: 0.24; elongation: 0.56; rugosity: 0.87; regularity: 1.07; and shape factor: 1.41. Different animals showed differences in their morphometric values. When we compared the values from different fractions, only two samples showed differences in morphometric parameter values and four samples showed differences in shape parameters. Multivariate analysis allowed the size classification of the cells into three classes and five classes of shapes. The distribution of classes among fractions showed no differences. Despite the individual morphometric differences observed in some fractions, the characteristics of the sperm head morphometry can be considered constant along the ejaculatory period in the llama.     

Activation of Mouse Oocytes Following Intracytoplasmic Injection of Chicken Sperm Extract

The cytosolic factor from the sperm of a wide variety of species has been reported to induce [Ca²⁺]i increase and/or activation in oocytes. Although many species have been studied, there is no data available on the chicken sperm factor responsible for activation of oocytes. This study was aimed to verify the activation of mouse oocyte by intracytoplasmic injection of chicken sperm extract (CSE). Survival rate of oocytes without rupture or lytic degeneration following injection was greater than 80% regardless of the presence or absence of CSE. Among the intact oocytes, activation rates following injection were 27.5% (11 of 40) in the sham operation group. Injection of CSE resulted in significantly higher activation rates in the 1/10 dilution group (57.8%, 26 of 45) compared with the sham operation group (p = 0.0045). Calculated amount of injected sperm extract of 1/10 dilution of CSE was equivalent of 12 chicken sperm. When the 1/10 diluted mouse sperm extract (MSE) was injected, survival rate of injected oocytes and activation rate of the survived oocytes was 82% (41 of 50) and 78% (32 of 41), respectively. Activation rate of MSE‐injected oocytes was a little but significantly higher than that of CSE‐injected oocytes (p < 0.037). In conclusion, it suggests that CSE can activate the mouse oocyte and that oocyte activating sperm factor(s) may be common between avian and mammal.     

Effects of Glucose Concentration on in vitro Fertilization in BALB/c Mice

BALB/c mice are widely used in genetic, tumour and immunological studies. However, the mice demonstrate a lower reproduction rate, low fertility and small litters, because of their highly genetic homozygoisty. Based on in vitro fertilization (IVF), a routine technique for biomedical studies, it is worth to evaluate the effects to BALB/c mice on IVF efficiency. In order to test the genetic factor affecting the IVF efficiency of BALB/c, four reciprocal IVF tests of BALB/cByJ and FVB/NCrl mice were performed. The results showed that the average fertility of IVF sponsored by FVB/NCrl spermatozoa was 69.6%, but only 12.1% was obtained from BALB/cByJ strain. Effect of glucose contained in the culture medium to the IVF efficiency of BALB/cByJ was also evaluated. The results showed that the fertility of BALB/cByJ spermatozoa incubated with 0, 2.7, 5.5, 11.1 and 22.2 mm of glucose in the TYH medium were 6.8, 9.9, 13.9, 32.7 and 22.2%, respectively. It is showed that IVF efficiency of BALB/cByJ spermatozoa could be improved depending on the concentration of glucose in the IVF medium. According to the results, it is beleived that lower IVF of BALB/cByJ mice might be due to the genetic defect in spermatozoa and increasing glucose in the IVF medium which significantly affect the IVF efficiency of BALB/cByl via activating the spermatozoa.     

Prolactin,Androstenedione and IGF1 Serum Concentrations During Induced Follicular Growth by eCG Administration in the Bitch

The oestrus cycle in the domestic bitch, a monoestrous species, differs considerably from that of other veterinary domestic animals species. In the bitch the combined use of eCG and hCG is effective to induce oestrus predictably and safely (Stornelli et al., Theriogenology, 78, 2012 and 1056). Although several studies were done to describe the hormonal changes during the canine oestrus cycle, to our knowledge none was done to describe the hormonal changes during induced follicular growth after the administration of eCG. The aim of this work was to study prolactin (PRL), insulin‐like growth factor (IGF1) and androstenedione (ANDR) serum concentrations during follicular growth induced by a single dose of eCG administered to late anoestrous bitches. PRL and ANDR concentrations were lower before than after eCG TRT (before eCG vs pro‐oestrus, oestrus and dioestrus; 4.3 ± 1.8 ng/ml vs 6.5 ± 1.6 ng/ml, p < 0.05; 0.08 ± 0.2 ng/ml vs 0.42 ± 0.16 ng/ml, p < 0.05). Conversely, IGF1 concentrations were similar before and after eCG TRT (286.0 ng/ml ±32.2, p > 0.53). Additionally, PRL concentrations were similar before oestrus compared to during oestrus and dioestrus (6.9 ± 1.7 ng/ml, p > 0.19). Furthermore, IGF1 concentrations were higher before and during oestrus compared to first day of dioestrus (286.1 ± 29.8vs 200.4 ± 29.2 ng/ml, p < 0.01). On the contrary, ANDR concentrations were lower before and during oestrus compared to first day of diestrum (0.35 ± 0.17 ng/ml and 0.38 ± 0.15 vs 0.68 ± 0.17 ng/ml, p < 0.05). These results show that treatment with a single injection of 50 IU/kg of eCG in late anoestrous bitches successfully induced changes in follicular growth which were paralleled with changes in PRL, IGF1 and ANDR serum concentration similar to those occurring during a normally occurring oestrous cycle. In addition, our results suggest that IGF1 in the bitch could play an important role in ovarian folliculogenesis.     

Seasonal Changes in Testicular Cell Morphology in Domestic Male Cats (Felis catus)

The aim of this study was to asses the variation in the morphology of the seminal epithelium in relation to natural photoperiod in male cats. Tom cats (n = 240) were castrated every other week throughout the year. Each testis was fixed in Bouin's solution and cut into sections. The percentage of tubules with round spermatids (RS), elongated spermatids (ES), tailed spermatids (TS), mature spermatids (MS) and the number of Sertoli cells (SC) and Leydig cells (LC) were recorded in each sample. Testicles from males during short days (SHD) had a higher percentage of tubules with RS and ES compared to testicles from males during long days (LHD, 31.3 ± 0.6 vs 2.1 ± 0.6%, p < 0.001; 30.9 ± 0.7 vs 11.0 ± 0.7%, p < 0.001). Conversely, testicles from males during SHD had a lower percentage of tubules with TS and MS compared to testicles from males during LHD (24.5 ± 0.8 vs 29.7 ± 0.8%, p < 0.01; 13.1 ± 1.2 vs 57.0 ± 1.2%, p < 0.01). Furthermore, testicles from males during SHD had a higher number of SC and lower number of LC compared to testicles from males during LHD (11.4 ± 0.1 vs 8.0 ± 0.1%, p < 0.01; 19.2 ± 1.0 vs 38.0 ± 1.0%, p < 0.01). In conclusion, there are seasonal changes in testis cell morphology in the tom which may be related to seasonal sperm production.     

Expression of Occludin in Canine Testis and Epididymis

Tight junctions (TJ) in inter-Sertoli junctional areas and epididymal epithelia are important for the formation of blood-testis barrier (BTB) and blood-epididymal barrier (BEB). In this study, the expression of occludin, an integral member of the TJ, was verified in canine testis and epididymis. Both low molecular weight (MW) (25-28 kDa) forms as well as high MW (68-72 kDa) forms of occludin were detected in the testis and epididymis using Western blot. The relative amount of the high MW forms of occludin vs low MW forms was higher in the testis than in the epididymis. Some difference in the composition of different MW forms of occludin was found along the segments of epididymis, suggesting the possible correlation between cellular composition of occludin proteins and paracellular permeability of epithelia along the epididymal tubule. In the testis, intense occludin immunoreactivity was found in the basally located inter-Sertoli junctional area. Diffused immunoreactivity of occludin was also found in the cytoplasm of Sertoli cells. A similar pattern of zonula occludens-1 immunoreactivity was found in the cytoplasm of Sertoli cells, suggesting that occludin was not confined to the inter-Sertoli junctional areas and that subcellular localization of occludin in the Sertoli cells was dynamically regulated during spermatogenesis in canine testis. In the epididymis weak immunoreactivity was found in the apical sides and cytoplasm of epithelial cells.     

Current attitudes to,and use of,peri-operative analgesia in dogs and cats by veterinarians in New Zealand

AIM: To investigate the attitudes of veterinary practitioners in New Zealand to pain and analgesia, and their use of analgesic drugs, in dogs and cats.

METHODS: A questionnaire posted to 1,200 practising veterinarians was used to gather information about the use of analgesia in dogs and cats, assessment of pain, attitudes to pain relief, analgesic drugs and procedures used, factors affecting choice of analgesic agent, and veterinary demographics, continuing education and staffing.

RESULTS: Three hundred and twenty questionnaires with useable data were returned, a response rate of 28%. Male and female veterinarians were evenly represented. The analgesic agents most commonly used were morphine (opioids) and carprofen (a non-steroidal anti-inflammatory drug; NSAID). Use of peri-operative pain relief ranged from 50% for castration of cats to 91% for fracture repair in dogs. For most procedures, female veterinarians scored pain at a significantly higher level than their male colleagues. Fifty-eight percent of respondents considered their knowledge in the area of assessment and treatment of pain was adequate.

CONCLUSIONS: This survey was considered representative of veterinarians working in companion animal practice in New Zealand. Results indicated a relatively high use of peri-operative analgesia, including both pre-emptive and multi-modal analge- sia, in cats and dogs, although there was still some disparity between the perception of how painful a procedure was and the consequent use of pain relief.

CLINICAL RELEVANCE: The establishment of current attitudes and practices indicates to practising veterinarians how their own use of analgesics compares with that of their colleagues. It also provides information to educators on potential areas of focus, given that 42% of respondents felt their knowledge in the area of assessment and treatment of pain was inadequate.