Análise histomorfométrica do útero na espécie canina do nascimento aos seis meses de idade / Histomorphometric analysis of the uterus in dogs from birth to six months of age

Objetivou-se caracterizar para a espécie canina o desenvolvimento histológico uterino do nascimento aos seis meses de idade. Foram analisados úteros (n=32) de animais com idades entre um e 180 dias pós-nascimento (DPN), distribuídos em oito grupos: G1-1 DPN (1 Yorkshire Terrier, 1 Poodle, 2 Sem Raça Definida (SRD)/peso médio (Pm)=190g), G2-15 DPN (2 Yorkshire Terrier e 2 SRD/Pm=354g), G3-30 DPN (1 Rottweiler, 1 Poodle, 2 SRD/Pm=985g), G4-45 DPN (1 Poodle, 3 SRD/Pm=1,1kg), G5-60 DPN (1 Yorkshire Terrier, 1 Poodle e 2 SRD/Pm=1,4kg), G6-90 DPN (4 SRD/Pm=2,8kg), G7-120 DPN (1 Poodle e 3 SRD/Pm=6,6kg) e G8-180 DPN (1 Rottweiler, 1 Poodle e 2 SRD/Pm=11kg). A análise histológica constituiu de mensurações das espessuras (µm) da parede uterina, miométrio, endométrio, assim como diâmetro (µm) e número de glândulas endometriais. A análise estatística demonstrou estabilidade e homogeneidade nas estruturas avaliadas com coeficientes de variação baixos (<10%). Observou-se: útero com um DPN: epitélio com células cúbicas simples, miométrio rudimentar, presença de perimétrio e ausência de glândulas endometriais; aos 15 DPN: desenvolvimento de estrutura primordial de formação das glândulas endometriais; dos 30 aos 45 DPN: endométrio e glândulas endometriais simples; aos 60 DPN: glândulas endometriais em lâmina própria com ramificações e discreto pregueamento endometrial; de 90 a 180 DPN: todas as estruturas uterinas apresentaram histoarquitetura de um animal maduro. Todas as variáveis analisadas apresentaram correlação positiva com a idade pós-nascimento (R2≥72,2%). Conclui-se que o útero da cadela desenvolve-se continuamente do nascimento à 180 dias de vida e que apresenta-se desprovido de glândulas endometriais ao nascimento. As primeiras glândulas endometriais são observadas aos 15 dias de vida e apresenta conformação histológica de um animal adulto após 60 dias do nascimento.

This study aimed to characterize canine species' uterine histological development from birth to six months of age. Uteri (n = 32) of animals aged between one and 180 days postnatal (PND), distributed into eight groups were analyzed: G1-1 PND (1 Yorkshire Terrier, Poodle 1, 2 Mixed Breed (MB)/Medium weight (Mw) = 190g), G2-15 PND (2 Yorkshire Terrier and 2 MB/Mw = 354g), G3-30 PND (1 Rottweiler, 1 Poodle, 2 MB/Mw = 985g), G4-45 PND (1 Poodle, 3 MB/Mw = 1.1kg), G5-60 PND (1 Yorkshire Terrier, Poodle 1 and 2 MB/Mw = 1.4kg), G6-90 PND (4 MB/Mw = 2.8kg), G7-120 PND (1 Poodle and 3 MB/Mw = 6.6kg) and G8-180 PND (1 Rottweiler, Poodle 1 and 2 MB/Mw = 11kg). Histological examination consisted of thickness measurement (µm) of the uterine wall, myometrium, endometrium, as well as diameter (µm) and number of endometrial glands. Statistical analysis showed stability and uniformity in the evaluated structures with low coefficients of variation (<10%). We observed: uterus with one PND: simple cubic epithelium cells, rudimentary myometrium, perimetrium presence and absence of endometrial glands; at 15 PND: development of primordial structure formation of endometrial glands; from 30 to 45 PND: simple endometrium and endometrial glands; PND 60: endometrial glands in the lamina propria with branches and discreet endometrial pleating; 90-180 PND: uterine all structures presented histoarchitecture of a mature animal. All variables were positively correlated with postnatal age (R2 ≥ 72.2%). It is concluded that the uterus of the bitch continuously evolves from birth to 180 days old and is presented devoid of endometrial glands at birth. The first endometrial glands are observed at 15 days of life and present histological conformation to an adult 60 days after birth.

A commercial box for dog semen transport: What happens inside when the environmental temperature is increasing?

Environmental temperatures may influence the temperature inside commercial transport boxes during semen shipment and thereby storage conditions of diluted dog semen. To evaluate the temperature changes inside boxes and their influence on sperm quality, split semen samples (n=8) were placed in Neopor boxes(®) exposed for 48h to room temperature (RT) (Box 1), 40°C for 6h and then kept at RT (Box 2) or 40°C (Box 3). A fourth subsample was kept at 4-5°C in a refrigerator (control). Inside Box 1 temperature initially decreased to <3°C before it stabilized at 7-8°C, while in Box 2 no decrease occurred and temperature was at 7-8°C for 48 h. Temperature inside Box 3 was at 14-15°C for 24h and, thereafter, increased to 36.1°C. Analysis of sperm motility (CASA) and viability (PI and FITC-PNA) after 24 and 48 h revealed marked sensitivity of dog spermatozoa to temperature fluctuations (Box 1). A constant storage temperature of 7-8°C (Box 2) provided the most desirable semen quality in terms of motility, viability, as well as osmotic resistance when samples were stored for 48 h. Furthermore, results indicate that during 24h preservation a storage temperature of 14-16°C may provide optimum conditions for maintenance of sperm viability and function. An increase of the inside temperature to >30°C (Box 3) resulted in an almost complete loss in sperm integrity. In conclusion, results suggest a revision of current recommendations for storage temperature of diluted dog semen. Boxes for semen transport should be prepared depending on the expected environmental temperatures.

Efficient association between PGF2α and methyl 4-hydroxybenzoate sex pheromone prior to electroejaculation in dogs.

Electroejaculation is a technique that can be used to collect semen from canines, but its use with this group of animals is restricted by low success rate and low semen quality. Here, we evaluated whether pharmacological and sexual sensory stimuli, which may affect ejaculation, can increase electroejaculation efficiency and improve ejaculate quality. We worked with 20 dogs of mixed breed weighing between 5.3 and 22.2 kg, divided into two groups. Both groups were exposed to a spayed female for 10 min, but in the second group, the same spayed female had her vagina impregnated with methyl 4-hydroxybenzoate synthetic pheromone for 10 min and after receiving dinoprost tromethamine IM, 0.1 mg/kg. After stimulation, all dogs were chemically restrained with ketamine, 8 mg/kg, IM; and xylazine, 1 mg/kg, IM, and subjected to electroejaculation protocol. We obtained 100% of antegrade ejaculate in treatments when the spayed female had her vagina impregnated with pheromone and 80% when she did not. Sperm motility was significantly different (p < 0.05) between controls and the test group (10.1 ± 4.5 and 43.0 ± 8.3, respectively). We concluded that the adopted electroejaculation protocol was efficient and that the PGF2α associated with sexual sensory stimulation can improve semen quality in dogs undergoing the procedure.

Effect of daily food supplementation with essential fatty acids on canine semen quality.

Polyunsaturated fatty acids are important membrane components that influence membrane integrity and fluidity. In the present study, the effect of oral supplementation for 60 days with essential fatty acids (omega 3, 6 and 9) and vitamin E on canine semen quality was evaluated. Sixteen dogs were selected for the experiment; eight were used as the control group and eight received the fatty acid supplemented diet for 60 days. Semen samples were taken every 15 days during the entire experimental period and were analyzed for volume (ml), motility (%), vigour (0-5), concentration (x10(6)/ml), morphology of spermatozoa (%), plasma membrane integrity (%; using the hyposmotic swelling test) and thermoresistance (motility and vigour after 4 h at 38 degrees C). We concluded that, daily supplementation with omega 3, omega 6 and omega 9 fatty acids, together with vitamin E, for a period of 60 days, significantly increased the semen volume of the treated group after 15 days of supplementation; the vigour and concentration of spermatozoa were superior after the first month of supplementation, while the percentage of morphologically abnormal spermatozoa decreased and the cells were protected against thermal stress.

Efeito da centrifugação sobre a qualidade do sêmen canino / Effect of centrifugation on quality of canine semen

Avaliou-se o efeito da centrifugação sobre a viabilidade do sêmen canino e compararam-se três meios de diluição pré-centrifugação. Utilizaram-se 10 ejaculados completos de 10 cães que, após a avaliação inicial, foram divididos em quatro porções (grupos). Uma das amostras, não centrifugada, formou o grupo-controle; as outras foram diluídas em três diferentes meios e centrifugadas a 800 x g por 15 minutos, formando os grupos: CPSA - constituído por sêmen centrifugado em plasma seminal autólogo; CLG - sêmen centrifugado em meio à base de leite desnatado e glicose (LG); e CPer- sêmen centrifugado em gradientes de Percoll (45 por cento e 90 por cento). Após a centrifugação e a eliminação do sobrenadante, procedeu-se à ressuspensão de todas as porções do ejaculado em LG e à imediata avaliação quanto à motilidade, vigor, aglutinação espermática e integridade das membranas espermáticas. Todas as suspensões foram, então, incubadas a 37ºC por 30 minutos e reavaliadas. O processo de centrifugação não causou danos aos espermatozoides e a centrifugação em meio LG melhorou a viabilidade espermática.

The effect of the centrifugation process on canine sperm viability was evaluated using three different precentrifugation extenders in the process. After an initial evaluation, ten complete ejaculates from ten dogs were used and subdivided in four groups. One sample of semen was not centrifuged and was used as control and the remaining samples of semen were diluted in three different extenders and centrifuged at 800 x g per 15 minutes, performing groups: CPSA- centrifuged in autologous seminal fluid, CLGcentrifuged in skim milk plus glucose extender (LG), and CPer- centrifuged under Percoll gradient (45 percent and 90 percent). After centrifugation, the resulting pellets were diluted in LG and evaluated for motility, viability, sperm agglutination, and spermatic membrane integrity. All the samples were incubated at 37ºC for 30 minutes and the evaluations were performed again. Centrifugation procedures did not induce damage to canine spermatozoa and samples centrifuged and diluted in skim milk plus glucose extender remained with better viability.

Quantity and quality of oocytes recovered from donor bitches of different ages.

In vitro studies that use isolated oocytes benefit from the ability to harvest oocytes of excellent morphological quality in sufficient numbers to allow the replicability of techniques and experiments. The objective of the present study was to verify the effect of the age of the donor bitch on the quantity and quality of oocytes recovered from isolated ovaries, using the slicing technique. Ten bitches (45 days to 13 years) were ovariohysterectomized, and the ovaries were placed in phosphate buffered saline (PBS), supplemented with bovine fetal serum (5%), and oocyte-cumulus-complexes (OCCs) were obtained by slicing the ovarian tissue. The OCSs were classified morphologically as Degree I (DI, best), Degree II (DII) and degenerated. A total of 427 oocytes were acquired, including 81, 109 and 237 that were graded as DI, DII and degenerated, respectively. Slicing yielded no OCS from animals < 2 months of age. In senile (> 9 years) bitches, bitches, there were more oocytes per bitch, compared to adult (2-6.5 years) bitches, but fewer DI oocytes, and more DII and degenerate oocytes. We inferred that using donors that were post-pubertal but not senile, would assure the recovery of high-quality oocytes by the slicing method. Additional studies are required to assess the quality of oocytes collected from pre-pubertal versus post-pubertal bitches < 2 years of age.