Rafael Gianella Mondadori

Intratesticular hypertonic sodium chloride solution treatment as a method of chemical castration in cattle

Castration of male calves is necessary for trading to facilitate handling and prevent reproduction. However, some methods of castration are traumatic and lead to economic losses because of infection and myiasis. The objective of the present study was to evaluate the efficiency of intratesticular injection (ITI) of hypertonic sodium chloride (NaCl; 20%) solution in male calf castration during the first weeks of life. Forty male calves were allocated to one of the following experimental groups: negative control-surgically castrated immediately after birth; positive control -intact males; G1-ITI from 1- to 5-day old; G2-ITI from 15- to 20-day old; and G3-ITI from 25- to 30-day old. Intratesticular injection induced coagulative necrosis of Leydig cells and seminiferous tubules leading to extensive fibrosis. Testosterone secretion and testicular development were severely impaired in 12-month-old animals from G1 and G2 groups (P<0.05), in which no testicular structure and sperm cells were observed during breeding soundness evaluation. Rectal and scrotal temperatures were not affected by different procedures. In conclusion, ITI of hypertonic NaCl solution induces sterility and completely suppresses testosterone secretion when performed during the first 20 days of life.

Períodos de digestão enzimática para o resgate de folículos pré-antrais em ovários de fetos bovinos

O presente experimento foi delineado com o objetivo de determinar a influencia de diferentes periodos de digestao enzimatica nos ovarios de fetos bovinos, sobre o numero total de foliculos pre-antrais resgatados e o numero de foliculos resgatados em cada estagio de desenvolvimento. A presenca da membrana basal, envolvendo o complexo formado pelas celulas da granulosa e o oocito, foi observada atraves de um estudo histologico, o que permitiu avaliar a integridade morfologica dos foliculos pre-antrais isolados. Para isso, ovarios de fetos bovinos, entre 150 e 270 dias de gestacao, foram obtidos em frigorifico. No laboratorio, os ovarios foram seccionados em varios fragmentos com uma tesoura cirurgica e dissociados com pipetas de Pasteur, com diâmetros aproximados de 1000 e 500mm. Apos este processo, os fragmentos foram submetidos a digestao enzimatica com colagenase em uma concentracao de 200UI/ml de TCM199 modificado, por periodos de 20, 30 ou 40 minutos. Os resultados obtidos com esta tecnica permitiram determinar que o numero de foliculos pre-antrais isolados, assim como, os estagios de desen- volvimento em que esses foliculos encontravam-se no momento do isolamento, sao similares nos tres periodos de incubacao enzimatica. A estrutura morfologica desses foliculos, formada pelo oocito, celulas da granulosa e membrana basal, manteve-se intacta apos o isolamento nos tres periodos de digestao enzimatica.

Selection of porcine oocytes in vitro through brilliant cresyl blue staining in distinct incubation media.

Staining with brilliant cresyl blue (BCB) may be used for oocyte selection, but BCB staining itself and the most commonly used selection medium (DMPBS) may compromise the development of porcine oocytes in vitro. This study evaluated DNA fragmentation, nuclear maturation, the area of migration of cortical granules (CG) and embryo development for stained (BCB+) and unstained (BCB-) oocytes incubated in DMPBS and in a modified medium (ReproPel) tested for the first time. Unexposed (UN), BCB+ and BCB- oocytes were incubated composing six groups: DMPBS/UN; DMPBS/BCB+; DMPBS/BCB-; ReproPel/UN; ReproPel/BCB+; and ReproPel/BCB-. There were more BCB+ oocytes in ReproPel than in DMPBS (P < 0.05). The DNA fragmentation was evaluated for oocytes in DMPBS/BCB+, DMPBS/BCB-, ReproPel/BCB+, ReproPel/BCB- and in porcine follicular fluid (control). The frequency of oocytes with no DNA fragmentation was greatest (64.6%) in DMPBS/BCB+ and lowest in ReproPel/BCB+ and ReproPel/BCB- (26.8 and 34.1%, respectively) (P < 0.05). Nuclear maturation rates were greater (P < 0.05) for DMPBS/BCB+ (63.1%), ReproPel/UN (55.1%) and ReproPel/BCB+ (50.2%) than for DMPBS/UN (40.8%) and ReproPel/BCB- (35.5%). The area of CG was greater (P < 0.05) for ReproPel/BCB- (80.7%) and DMPBS/UN (77.6%) than for ReproPel/UN (34.7%). Cleavage rates for DMPBS/BCB+ and ReproPel/BCB+ were greater than for DMPBS/UN (P < 0.05). Blastocyst development rates were greatest (P < 0.05) for ReproPel/UN and ReproPel/BCB+. In both media, BCB staining was apparently unable to select competent oocytes, which likely occurred due to toxicity. Despite the similar nuclear maturation and area of CG compared with DMPBS, oocytes selected in ReproPel presented impaired DNA integrity.

The Use of Antibiotics in Cryopreservation of Ram Sperm

Antibiotics are used in extenders for sperm cryopreservation, however no specific regulation has been established for ram sperm. This study evaluates the effects of antibiotics in ram sperm extenders for bacterial control and its effect on sperm viability. Sperm from five rams was collected, cooled and frozen using extenders with the following antibiotics: 100,000 IU/mL penicillin and 100 µg/mL streptomycin (PES); 500 µg/mL gentamycin, 100 µg/mL tylosin, 300 µg/mL lincomycin and 600 µg/mL spectinomycin (GTLS); 50 µg/mL ceftiofur sodium (CEF); and 1,000 µg/mL enrofloxacin (ENR). Bacillus sp., Corynebacterium sp., Klebsiella sp. and Staphylococcus sp. were isolated from fresh sperm and perpetual area. For cooled sperm, the antibiotic PES, GTLS and ENR were able to reduce the number of colony forming units per mL (CFU/mL), however, CEF was inefficient in all tested concentrations. Motility of cooled sperm was reduced when GTLS and ENR were used (P 0.05), however, motility was reduced with ENR treatment at doses greater than 50% (P < 0.05). The antibiogram revealed resistance of Staphylococcus sp . and Klebsiella sp. to the penicillin/streptomycin association and to tylosin. Both PES and GTLS were able to control bacterial growth on cooled sperm. However, attention should be given to the antibiotics added to the extenders, which may impair sperm motility.

Fecundação e clivagem após a ativação da proteína quinase C durante a maturação de oócitos bovinos

In the present study the pronucleus formation, cleavage and protein profile after protein kinase C (PK-C) activation were evaluated during bovine oocyte maturation. A total number of 1936 bovine cumulus-oocyte complexes (COCs) were randomly distributed in 4 treatments, and matured with PMA (100nM phorbol 12-myristate 13-acetate, PK-C activator), 4a-PDD (100nM de 4a-phorbol 12,13-didecanoetate, phobol ester that do not bind on PK-C, phorbol ester control), ECS (10% of estrus cow serum, positive control) and in a negative control (basic maturation media for all treatments, except on ECS group where the polyvinyl alcohol was not added). The COCs were kept in presence of PMA for 1, 4 or 7 hours and then transferred to basic maturation media until the 24 hours maturation period was completed. The PK-C stimulation in these periods did not alter the pronucleus formation but caused a decrease in the zygotes cleavage rate. The 4a-PDD group results showed that the alterations in PMA group were caused by PK-C activation and not by direct phorbol ester action on the cell. The protein composition analysis showed an additional protein band on ECS group around 74 kilo Daltons (kDa). Altogether, these results with the preliminary Western Blot analysis, indicate an important role of PK-C on bovine oocyte maturation, fertilization and embryo cleavage.

Histological description of Cerdocyon thous (Linnaeus, 1766) respiratory system

Rocha M.P., Nunes A.P., Minello L.F., Cruz L.A.X., Albano A.P.N. & Mondadori R.G. 2017. Histological description of Cerdocyon thous (Linnaeus, 1766) respiratory system. Pesquisa Veterinária Brasileira 37(5):531-535. Faculdade de Medicina, Instituto de Biologia, Departamento de Morfologia, Universidade Federal de Pelotas, Av. Duque de Caxias 250, Fragata, Pelotas, RS 96030-000, Brazil. E-mail: marlapi@yahoo.com.br The massive agricultural expansion converted the Cerdocyon thous, a South American native predator, in vulnerable specie. Basic data, such as histological description, are important to raise awareness on animal species, helping on preservation strategies. Considering the difficult in obtain samples, as the euthanasia of wild animals for this purpose is not allowed, data on histology are very scarce or inexistent. The objective of this paper was to provide a detailed histological description of the trachea and bronchial tree of the crab-eating fox Cerdocyon thous (Linnaeus, 1766). The specimens (one adult male and one adult female) used were provided by the Federal University of Pelotas (Pelotas, RS, Brazil) Rehabilitation Center of Wild Fauna (NURFS). Tissue samples were fixed in 10% formalin and included in paraffin. After slicing, samples were stained with HE (hematoxylin and eosin), PAS (periodic acid–Schiff) and resorcin fuchsin. Trachea had an average diameter of 7.87mm, and approximately 57% of the mucosa ciliated pseudo-stratified columnar epithelium was composed of goblet cells, mostly in the dorsal region. Bronchia and bronchioles had a mucosal fold with higher number of goblet cells. Using all these techniques there is no great remarkable differences from C. thous trachea and lung, when compared with the previous described structures for carnivores and most mammals, except for the goblet cells “regionalization”. Described results are important to understand the animal physiological and behavioral habits, allowing the development of preservation and protection strategies.

Effect of β-mercaptoetanol and cysteine on post-thawing quality and oxidative activity of ram sperm and on the viability of vitrified sheep embryos

The effects of β-mercaptoethanol (BME) and cysteine on the viability and oxidative activity of ram sperm after thawing and on development in vitro and viability of vitrified sheep embryos were evaluated. Ejaculates from four rams were pooled and extended, composing six treatments: no antioxidants; 2mM BME; 5mM BME; 2mM BME and 5mM cysteine; 5mM BME and 5mM cysteine; and 5mM cysteine. Sperm motility, membrane and acrosome integrity, mitochondrial functionality, production of reactive oxygen species and total antioxidant capacity were similar across treatments (P>0.05). A medium with no antioxidant presented cleavage and blastocyst development rates (60.3% and 33.6%, respectively) similar (P>0.05) to those of a medium with 50μM BME and 600μM cysteine (64.3% and 36.6%, respectively). Post-thawing viability of vitrified embryos was similar between media (P>0.05). Cysteine and BME had no influence on the post-thawing viability and oxidative activity of ram sperm and on the viability of vitrified sheep embryos.