Successful artificial insemination (AI) in the koala using neat and extended semen collected by electroejaculation (EE)

Presently AI in the koala has been based on the insemination of fresh undiluted semen collected with an artificial vagina (1). While this approach has been extremely successful, further refinement and implementation of AI for use with cryopreserved semen will require protocols that incorporate diluted semen collected by EE. Recent studies have shown that koala semen is likely to have an "ovulation factor" such that over-dilution may result in ovulation failure (2). The current study determined whether AI of EEed neat and/or diluted semen was capable of inducing a luteal phase and/or resulted in the production of pouch young. All koalas were inseminated in the breeding season between day 2 and 5 of oestrus and subsequently monitored for evidence of parturition (day 35) and return of oestrus. Successful induction of a luteal phase was based on evidence of an elevated progesterone concentration 28 days after insemination (2). All semen samples were collected by EE and seminal characteristics recorded (3). The diluent used for semen extension was Tris-citrate glucose (TCG) which contained antibiotics but no egg yolk (4). AI was conducted on conscious koalas using a "Cook koala insemination catheter" and a glass rod used to mimic penile thrusting (1). Three insemination treatments were used; (A) 1mL of undiluted semen (n = 9); (B) 2mL of 1:1 diluted semen (n = 9); and (C) 1 mL of 1:1 diluted semen (n = 9). The results of the AI trial are shown in Table 1. This study has shown that it is possible to use both neat and diluted semen (1:1; 1 or 2 mL) to successfully produce koala offspring at conception rates similar to those achieved following natural mating. Interestingly, dilution of semen had no apparent detrimental effect on induction of a luteal phase following AI.

Collection and evaluation of semen from the three-toed sloth (Bradypus tridactylus)

Sloths (Bradypus sp.) are extremely sensitive animals that suffer with the destruction and fragmentation of forests. They present a low population growth rate and need to be further studied for the preservation of the specie. Thus, the aim of this study was to establish an efficient semen collection protocol as well as characterize sperm concentration, motility and morphology in order to contribute with information about the reproductive traits of this specie, which has never been described in the literature before. For that, nine Bradypus tridactylus males were captured during the wet season and six during the dry season, in Manaus (AM), located in the north region of Brazil, semen was collected by electroejaculation with shocks given in sequences of progressive intensities (minimum 20 mA and maximum 60 mA). All animals ejaculated small volumes of semen and in some of them, the volume ejaculated was not enough for a complete spermiogram. Physical characteristics observed on the collections of the wet season were different from those seen in the specimen collected in the dry season. Motility an vigor was very low and did not show forward progression, only oscillatory movement. After Spermac stain, spermatozoa presented a wide variety of defects; however, the differences in morphology were not significant between seasons. The morphology assessed by scanning electron microscopy shows that the head in both groups could be elongated, short or could have a base narrower than the apex and the midpiece narrowed abruptly, forming a nip in its transition to the tail. Although further studies are necessary to verify our preliminary findings concerning seasonal variation in sperm quality, these results demonstrate that semen can be safely collected from sloths by electroejaculation and provide the first reports of semen characteristics in this species. (C) 2008 Elsevier Ltd. All rights reserved.

Sperm morphological and morphometric evaluation in captive collared peccaries (Pecari tajacu)

The aim of this study was to compare different staining methods for the evaluation of sperm morphology by light microscopy and also to describe the morphometry of the entire sperm in collared peccaries (Pecari tajacu). Semen from 10 males was obtained by electroejaculation and evaluated for sperm motility, vigor, and concentration. Semen smears were prepared through three different staining methods: Bengal rose, brome-phenol blue, and eosin-nigrosin. Smears were evaluated under light microscopy and sperm morphologic alterations were determined in percentage. In addition, sperm morphometric analysis was conducted by light microscopy coupled to image analyzer software. The smears stained with Bengal Rose provide the best results for the visualization of the sperm tail, midpiece, and head. The use of eosin-nigrosin stain did not allow an adequate impregnation, and some sperm presented a few contrasts with the background. A higher incidence of bent coiled tails was verified in the use of brome-phenol blue staining (P<0.05). Through morphometric evaluation, it was observed that the tail occupies the greatest proportion (89%) of the sperm which presents a discretely elongated head. According to the results, the use of the Bengal Rose stain is recommended for the morphologic evaluation of the collared peccary sperm.

Changes in the fecal concentrations of cortisol and androgen metabolites in captive male jaguars (Panthera onca) in response to stress

In the present study we determined the efficacy of the measurement of fecal cortisol and androgen metabolite concentrations to monitor adrenal and testicular activity in the jaguar (Panthera onca). Three captive male jaguars were chemically restrained and electroejaculated once or twice within a period of two months. Fecal samples were collected daily for 5 days before and 5 days after the procedure and stored at -20ºC until extraction. Variations in the concentrations of cortisol and androgen metabolites before and after the procedure were determined by solid phase cortisol and testosterone radioimmunoassay and feces dry weight was determined by drying at 37ºC for 24 h under vacuum. On four occasions, fecal cortisol metabolite levels were elevated above baseline (307.8 ± 17.5 ng/g dry feces) in the first fecal sample collected after the procedure (100 to 350% above baseline). On one occasion, we did not detect any variation. Mean (± SEM) fecal androgen concentration did not change after chemical restraint and electroejaculation (before: 131.1 ± 26.7, after: 213.7 ± 43.6 ng/g dry feces). These data show that determination of fecal cortisol and androgen metabolites can be very useful for a noninvasive assessment of animal well-being and as a complement to behavioral, physiological, and pathological studies. It can also be useful for the study of the relationship between adrenal activity and reproductive performance in the jaguar.

Studies on the female reproductive physiology of the prawn, Macrobrachium idella (Hilgendorf, 1898)

’l‘he reproductive physiology of the female palaemonid prawn M. idella has been investigated by adopting a comprehensive approach to the problem. The major aspects of the study included investigations on breeding biology and process of oogenesis, variations in the biochemical components in relation to maturation, neuroendocrine relations and control over reproduction, and artificial insemination. The prawns used in the present study were procured from Vembanad Lake at Panavally village - a place nearly 20 km. away from Cochin. The studies were carried out using standard histological and biochemical methods. The modern technique of electroejaculation was adopted for extrusion of spermatophores in artificial insemination experiment.

Estudo preliminar da influência do perímetro escrotal sobre a libido em touros jovens da raça Nelore

O perímetro escrotal (PE) de 202 touros jovens da raça Nelore, sem experiência sexual prévia, classificados pela Associação Brasileira de Criadores de Zebu como superiores ou elite, foi medido aos 7, 12, 18 e 28 meses de idade, aproximadamente. Após a última medição, eles foram submetidos ao teste de libido e procedeu-se à colheita de sêmen por eletroejaculação, seguida de avaliação andrológica. As medidas do PE aos 7, 12, 18 e 28 meses foram de 18,30; 22,29; 27,54 e 33,26cm, respectivamente. Os coeficientes de correlação simples entre estas medidas variaram de 0,35 a 0,53. O coeficiente de correlação entre PE aos 28 meses e peso corporal na mesma idade foi de 0,43. A correlação entre a nota da libido e o perímetro escrotal medido aos l8 meses foi de 0,15 e entre aquela e o perímetro escrotal aos 28 meses foi de 0,13. Observou-se elevado crescimento do perímetro escrotal entre o 7º e 18º meses de idade. Utilizou-se o método de quadrados mínimos para analisar a libido, incluindo ano de nascimento como efeito fixo e como covariáveis os efeitos lineares da idade e peso no momento da avaliação e medidas de perímetro escrotal aos 7, l2, l8 e 28 meses. Cada covariável foi retida de forma seqüencial obtendo-se vários modelos de análises e as somas de quadrados foram decompostas de forma seqüencial e parcial. O ano de nascimento, a idade e o perímetro escrotal aos 18 meses de idade afetaram significativamente a libido. Os coeficientes de correlação entre a libido e concentração espermática, motilidade, peso e idade do animal no momento da avaliação foram de 0,34, 0,l6, 0,38 e 0,35, respectivamente.

Effects of scrotal insulation in Nellore bulls (Bos taurus indicus) on seminal quality and its relationship with in vitro fertilizing ability

The objectives of this study were to assess the effects of induced testicular degeneration in Bos taurus indicus (Nellore) bulls on changes in seminal characteristics and fertilizing ability of sperm. Four Nellore bulls (30-36-month-old, 500-550 kg) with good seminal quality (> 80% motile and morphologically normal sperm) had serotal insulation applied for 5 d. Semen was collected by electroejaculation and cryopreserved at a the pre-insulation moment, and 7, 14, and 21 d after insulation was removed. Gross motility, vigor of sperm movement (1-5), acrosome integrity, sperm morphology (phase-contrast microscopy), nuclear vacuoles and abnormal chromatin (Feulgen-stain) were determined after sperm preparations for in vitro fertilization (IVF). Prior to IVF, sperm were separated using a Percoll gradient (45% and 90%). Normal sperm decreased (P < 0.05) 14 and 21 d after insulation was removed. on 14 and 21 d, the incidence of head defects (9.7 +/- 0.6 and 17.0 +/- 0.8, respectively; mean +/- S.E.M) was higher (P < 0.05) in agreement with the incidence of nuclear vauoles (14.0 +/- 5.0 and 12.3 +/- 2.3) and abnormal chromatin (24.4 +/- 7.2 and 30.8 +/- 2.8). Although the frequency of cleaved oocytes decreased only on 21 d (P < 0.05), blastocyst rates were lower (P < 0.05) than pre-insulation on 14 and 21 d. In regression analyses, only nuclear vacuoles, head defects and intact acrosome accounted for differences in cleavage (R(2) = 0.38, 0.48, and 0.30, respectively) and blastocyst rates (R(2) = 0.35, 0.37, and 0.44). Abnormal chromatin was associated only with blastocyst rates (R(2) = 0.35). In conclusion, blastocyst rate was more sensitive than cleavage rate and the assessment of nuclear integrity is recommended to predict the fertilizing ability of bull sperm. (c) 2008 Elsevier B.V. All rights reserved.

Cryopreservation effects on domestic cat epididymal versus electroejaculated spermatozoa

Frozen-thawed epididymal spermatozoa have already been successfully used in artificial insemination in the domestic cat, proving to be a valuable resource for the reproduction of felid species, which are threatened with extinction. The aim of this study was to compare the effects of freezing and thawing on domestic cat semen collected by electroejaculation (EL) and from the epididymides (EP) and vasa deferentia. Ten adult cats were anesthetized, electroejaculated and immediately thereafter, orchiectomized. Epididymal spermatozoa were collected through the compression of caudae epididymidis and vasa deferentia. Spermatozoa were frozen-thawed following a single protocol. Sperm motility, sperm progressive status (0-5), plasma membrane integrity and morphology (light and transmission electron microscope) were assessed on two occasions, immediately after collection and after freezing and thawing. There were no significant differences between the electroejaculated and epididymal fresh or frozen-thawed spermatozoa for any of the variables. However, the incidence of acrosome defects after freezing and thawing increased by 19% based on light microscopy, whereas ultrastructural images revealed acrosome damages in most sperm cells. Since these acrosomal changes are known to affect sperm fertilising capacity, further studies are needed to optimize cryopreservation techniques for epididymal as well as electroejaculated domestic cat spermatozoa. (c) 2006 Elsevier B.V. All rights reserved.

Colheita de sêmen por eletroejaculação em cutia-parda (Dasyprocta azarae)

As cutias são animais que vivem nas Américas Central e do Sul, com potencial zootécnico e que despertam crescente interesse científico. O objetivo deste estudo foi estabelecer um padrão para colheita de sêmen por eletroejaculação em cutia-parda (Dasyprocta azarae). Foram utilizados machos adultos (n=4) anestesiados e submetidos a quatro séries de 20 estímulos eletroejaculatórios, com 3 segundos de duração cada, com uma intensidade de 2, 4, 6 e 8 volts, com dois minutos de intervalo entre cada série. A colheita de sêmen foi obtida em todos os animais, com ereção parcial com 2 volts, ereção evidente com 4 volts, ejaculação com 6 volts e exacerbação da glande e exteriorização das espículas penianas com 8 volts, observando-se espermatozoides em 100% das amostras. A técnica descrita para colheita de sêmen em cutia-parda foi eficaz.

Citologia aspirativa por agulha fina (CAAF), em testículo de onça pintada (Panthera onca), utilizada como ferramenta no diagnóstico de infertilidade

Técnicas de biópsia, caracterizadas pela remoção de segmentos de órgãos e tecidos para análise histopatológica, não são indicadas no auxílio diagnóstico de alterações testiculares para animais ameaçados de extinção, por não serem totalmente isentas de riscos. Neste sentido, é de grande interesse que se desenvolvam técnicas de biópsia testicular cada vez mais seguras e com o mínimo de conseqüências negativas. Com este intuito três onças pintadas (Panthera onca) foram submetidas a exames de Citologia Aspirativa por Agulha Fina (CAAF). Amostras foram obtidas através da punção aspirativa dos testículos, esfregaços foram confeccionados, corados com Panótico e analisados sob Microscopia Óptica. Simultaneamente foram realizadas coletas de sêmen para avaliação do volume, pH, concentração, motilidade, vigor e morfologia espermáticas. Quanto à avaliação espermática, os animais apresentaram valores semelhantes aos encontrados na literatura quanto ao volume, pH, motilidade, vigor e morfologia espermáticas. Quanto a concentração espermática os animais apresentaram valores abaixo dos encontrados na literatura. Nos exames de CAAF, todas as gerações de células germinativas foram identificadas, indicando espermatogênese normal em todos os animais, com exceção das espermátides finais duplas que ainda não foram relatadas como achados em punções testiculares de outras espécies, o que vem confirmar a elevada porcentagem de células teratológicas encontradas nesses animais. Desta forma, podemos concluir que a CAAF testicular é um método diagnóstico auxiliar importante na detecção de alterações testiculares em casos de sub ou infertilidade, podendo ser utilizados na rotina de investigação do trato reprodutivo masculino, quando o exame histopatológico, por ser um método altamente invasivo, é desaconselhável.

Use of cooled bull semen as a strategy to increase the pregnancy rate in fixed-time artificial insemination programs-case report

Semen cryopreservation is still considered suboptimal due to lower fertility when compared to fresh semen. The reasons for the loss of fertility are various and related to irreversible damage caused to the cells during the freeze-thaw process. An alternative to conventional cryopreservation represents the use of chilled bull semen, preventing the damage associated with freezing, thereby guaranteeing greater sperm viability. The aim of this study was to describe the use of cooled bull semen as a strategy to increase the pregnancy for Fixed-Time Artificial Insemination (FTAI) of Nellore (Bos indicus) cows. One ejaculate of a select Nellore bull obtained by electroejaculation was used; the semen sample was fractioned into two aliquots: one diluted in Botu-Bov® extender containing 6.4% glycerol for cryopreservation (BB-F, frozen group) and one diluted in the same extender, free from cryoprotectants and used for cooling (BB-C, cooled semen group). The samples in the BB-C group were chilled to 5°C using an isothermic box and maintained for 24 h prior to use. A total of 349 lactating Nellore cows (70-90 days after birth) were synchronized by the insertion of a progesterone releasing device (1.0 g) and estradiol benzoate (2.0 mg i.m.) on a random day of the estrous cycle (Day 0); FTAI was performed 44-48 h after the removal of the device. The pregnancy rates were 45.71 and 61.49% (P<0.05), respectively, for the cryopreserved or chilled bovine semen groups. In conclusion, the use of bull semen cooled for 24 h represents an alternative to conventionally cryopreserved semen, as determined by the increase the pregnancy per artificial insemination in bovine herds. © 2012 Science Publication.

Evaluation of semen characteristics of the species Mazama americana in captivity

In recent years the concept of genomic resource banks has grown as a way of maintaining the genetic variability of populations, while the quality of cryopreservation of the gametes determines the effectiveness of such banks. However, the absence of basic knowledge regarding the physiology of species and their semen characteristics hampers the establishment of reproductive biotechnologies. Thus, this paper aimed to determine certain physicochemical (volume, colour, appearance, pH and osmolarity) and microscopic characteristics (mass movement, motility, vigour, concentration, and sperm morphology and morphometry) of semen of the species Mazama americana. To achieve this, five males of the species were used, and three semen samples per buck (electroejaculation) were collected at intervals of 2 weeks. The volume, pH and osmolarity of the ejaculate were 0.39 ± 0.14 mL, 6.90 ± 0.74 and 297.74 ± 19.10 mOsm/kg, respectively, while the values obtained for mass movement, motility, vigour and concentration were 3.33 ± 0.82; 69.6 ± 8.92%; 3.53 ± 0.50, and 244.07 ± 98.65 × 107/mL, respectively. Regarding the colour of the ejaculate, five samples were classified as ivory, two as yellowish, two as whitish and six as white. Regarding appearance, seven samples were considered creamy and eight, milky. Morphology was analysed in a humid chamber under phase contrast microscopy and 73.50 ± 5.57% of cells presented normal morphology, 8.37 ± 3.15% presented major defects and 18.13 ± 6.46% presented minor defects. To determine sperm morphometry, an optical microscope (Leica DM 5000B) and the Leica Qwin image analyser program were used, resulting in 8.09 ± 0.40, 4.65 ± 0.30, 2.81 ± 0.44 and 30.25 ± 3.02 m for length, largest width, smallest width and area, respectively. Copyright © CSIRO 2013.

Seasonal effects on semen and testosterone in zebu and taurine bulls

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Efeito do meio diluidor e da dose inseminante sobre a congebilidade e fertilidade do sêmen bovino utilizado em programas de inseminação artificial em tempo-fixo(LATF)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Biometria testicular, eletroejaculação e características seminais de caititus, Tayassu tajacu Linnaeus, 1758 (Mammalia, Artiodactyla, Tayassuidae) mantidos em cativeiro na Amazônia Oriental

Estudos relacionados à obtenção e avaliação de sêmen de Tayassu tajacu são escassos, sendo necessárias pesquisas a respeito. Os objetivos do estudo foram avaliar a biometria testicular de caititus adultos cativos, testar a eficiência da eletroejaculação para obtenção de sêmen e avaliar suas características seminais ao longo do ano. Procedeu-se à eletroejaculação em oito animais adultos e as amostras de sêmen colhidas foram avaliadas quanto às características físicas e morfológicas. Os animais tinham testículo esquerdo com 3,8 ± 0,4 cm X 2,6 ± 0,3 cm e 2,3 ± 0,2 de consistência, e testículo direito com 3,8 ± 0,5 cm X 2,7 ± 0,3 cm e 2,3 ± 0,2 de consistência. A taxa de sucesso nas colheitas foi de 75,21%. O sêmen possuiu: volume 0,81 ± 0,86 mL, concentração 137,44 ± 153 x 106 sptz mL-1, pH 7,92 ± 0,73, motilidade 52,66 ± 28,79%, vigor 2,2 ± 0,8, integridade de membrana plasmática 55,84 ± 28,55%, defeitos maiores 22,87 ± 12,93%, defeitos menores 9,11 ± 5,88% e defeitos totais 31,52 ± 13,81%. Os animais apresentaram simetria testicular, a eletroejaculação se mostrou eficiente para a obtenção de ejaculados em caititus e as flutuações observadas na produção seminal não foram suficientes para caracterizá-los como animais de reprodução sazonal.