Mean Number Of Spermatozoa Research Articles

A simple method for repeated in vivo sperm collection from laboratory mice

PurposeMouse spermatozoa for archiving laboratory mice or for in vitro fertilization (IVF) are routinely obtained from the cauda epididymis of adult males sacrificed for this purpose. To avoid the death of the donor, we tested whether a precisely timed interruption of the mating act could be used for repeated sperm collection from laboratory mice.MethodsSperm donors (B6D2F1) were mated with a receptive female, and mating behavior was observed. The stud was separated from the female 1–2 s after the onset of the ejaculatory shudder. The ejected copulatory plug with the yellowish viscous ejaculate was carefully removed from the penile cup.ResultsA total of 80 ejaculates were successfully obtained from 100 ejaculations. The latency to first mount was 1.1 ± 1.1 min (mean ± SD) and to ejaculation 8.1 ± 4.7 min. The average number of mounts to ejaculation was 10.5 ± 5.8, and the mean number of spermatozoa per collected ejaculate was 1.86 ± 1.05 × 106. An average fertilization rate of 76% was observed after IVF.ConclusionsSeparating the stud from the female just before ejaculation is feasible, easy to learn, and requires no special equipment. The sperm count of collected ejaculates is lower than natural ejaculations, but higher than previous in vivo sperm collection methods achieved. We recommend this simple sperm collection method in mice, especially when the donor cannot be sacrificed and/or repeated sperm collection from the same animal is required for experimental purposes.

Morphology of the testes of animals during estrogen injection in the prenatal period

The aim of the study was to investigate the morphological changes in the testes of the offspring of white non-pedigreed laboratory mice when the synthetic oestrogen analogue synestrol was prenatally injected to the mother. After fertilization, the females were divided into 2 groups which received a single intramuscular injection of the synthetic oestrogen analogue synestrol on day E 11.5 of gestation at the same time of the day. The intact group was not exposed to any treatment. The experimental group was exposed to synestrol, a synthetic oestrogen analogue, as a 2% oil solution at a dose of 40 µg/kg (C-40). The results of morphometric analysis of testes of descendants of white outbred laboratory mice after prenatal single injection of synthetic oestrogen analogue synestrol in a dose of 40 µg/kg showed morphological changes in the organ parenchyma, manifested as decrease in mean number of Sertoli cells (C-40) 17.4±1.1 compared to intact group 20.8±1.9; decrease in mean number of spermatogonia (C-40) 24.4±1.1 compared to intact group, decrease in mean number of spermatozoa (C-40) 178.0±4.2 compared to intact group 196.6±5.3. There was observed a change in the endocrine apparatus of testes, expressed as a decrease in the mean area of Leydig cell nuclei in the intact group of 6,72±1,78. Prenatal effects of synestrol revealed in an experimental model, allow us to use it to search for means of postnatal developmental testicular dysfunction correction as well as to develop optimal doses of oestrogenic preparations during pregnancy.

Periovulatory oviductal fluid decreases sperm protein kinase A activity, tyrosine phosphorylation, and in vitro fertilization in pig.

Molecules from the female reproductive tract modulate capacitation and function of sperm cells in vivo. These molecules vary in a quantitative and qualitative manner throughout the estrous cycle. This work evaluates the effect of using various female reproductive fluids on capacitation and fertilization of pig spermatozoa in vitro. The effects of culturing spermatozoa in different fluids on the levels of sperm protein kinase A (pPKA), tyrosine phosphorylation, acrosome reaction, and in vitro fertilization (IVF) were evaluated. The fluids tested were as follows: oviductal fluid (OF) from five phases of the estrous cycle, namely early and late follicular (OF-EF, OF-LF), early and late luteal (OF-EL, OF-LL) and periovulatory (pOF), follicular fluid from medium-sized follicles, and secretions of cumulus-oocyte complexes (conditioned medium). The pPKAs and tyrosine phosphorylation were decreased by OF-EF, OF-LF, OF-EL, and pOF but not by follicular fluid and conditioned medium. OF-EF, OF-LF, and pOF also decreased the sperm acrosome reaction. Moreover, the effect of pOF on pPKAs and tyrosine phosphorylation was reversible. In in vitro fertilization, OF-EF, OF-LF, OF-EL, and pOF reduced the percentage of penetrated oocytes, the mean number of spermatozoa per penetrated oocyte, and increased monospermy. OF from follicular, early luteal, and periovulatory phases of the estrous cycle modulates the sperm protein phosphorylation as well as the acrosome reaction involved in capacitation and increases monospermic fertilization in in vitro fertilization. Our findings suggest that fluids from the female reproductive tract could be used as additives in porcine IVF systems to modulate sperm-oocyte interaction.

Sperm DNA fragmentation in men of different age

The study objective is to analyze the content of spermatozoa with single and double-stranded DNA breaks in different age groups.Materials and methods. The level of DNA fragmentation was studied in 300 ejaculate samples obtained from 266 sub- or infertile men. The group 1 included 150 samples obtained from 131 patients under the age of 45 (21–44 years), the group 2 included 150 samples obtained from 135 patients above the age of 45 (45–68 years). Mean ages were 34.8 ± 3.9 and 48.6 ± 3.1 years, respectively. The number of sperm with fragmented DNA was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method on ejaculate smears. The number of spermatozoa with >15 % of fragmented DNA was considered elevated. Standard semen analysis was performed in 117 and 97 men from the groups 1 and 2, respectively.Results. The number of sperm with fragmented DNA varied in ejaculated samples from 1.5 to 64.5 %. Mean number of sperm with DNA breaks in the group 1 (12.0 ± 6.0 %) was significantly lower than in the group 2 (16.1 ± 8.3 %, p <0.05). Mean sperm count in the ejaculate of the group 1 (267.0 ± 198.7 million) was significantly higher than in the group 2 (201.0 ± 162.9 million, p = 0.02).Conclusion. We revealed that in men over the age of 45 years, the percentage of spermatozoa with DNA fragmentation is higher than in men under 45 years of age, it may indirectly indicate an increased level of reactive oxygen species in the seminal plasma in older patients.

Electrostimulation is an effective and safe method for semen collection in medium-sized lizards

The development of safe and consistent semen collection protocols should be ensured to understand basic sperm parameters of a species. Electroejaculation has been hypothesized and tested to be a safe method to evaluate male reproductive potential in wild animals. However, little is known about semen collection protocols in lizards. Adjusting stimulation to species and body mass is important for efficient semen collection as well as for animal welfare. Tropidurus spinulosus is a good model to adapt electrostimulation; it is a medium-sized lizard species, males have semen during a long period and operative sex ratio is male-biased. We aimed to provide a thorough and safe method for collecting semen samples from this animal model by means of electrostimulation and characterize basic sperm parameters. Mature males of T. spinulosus were captured and their testicular volume was evaluated via portable ultrasound scanning. The lizards were electrostimulated by performing standardized series of stimuli. Semen was obtained successfully in 94% of the males. Samples were contamination-free. Mean sperm number of ejaculates was 2.1 ± 1.8 × 106 spermatozoids. The percentage of motile spermatozoa was 78% and sperm dynamic parameters were: VSL 37.26 ± 7.72 μ/s and VCL 84.26 ± 16.27 μ/s. We observed high variability in testicular volume among males; however, almost all the individuals had sperm. Electrostimulation using protocols adjusted to a medium-sized lizard was an effective semen collection method that allowed us to obtain semen samples with high motility (percentage of motile spermatozoa and sperm velocity).

Colloid centrifugation of fresh semen improves post-thaw quality of cryopreserved dromedary camel spermatozoa

Colloids have been successfully used in a number of species to improve sperm populations for IVF and for cryopreservation The usefulness of Single Layer Centrifugation (SLC) for freezing dromedary camel spermatozoa in two different extenders was evaluated by examining the motility, viability, acrosome status, DNA integrity, and ability of cryopreserved sperm to penetrate oocytes in vitro in a heterologus IVF system. Two ejaculates from each of five males were divided into four aliquots: two were processed by SLC (selected) while two were centrifuged without colloid (control). Pellets were cryopreserved in Green Buffer or INRA-96® containing 3% glycerol and evaluated at 0 and 1 h post thawed. The SLC improved post-thaw total and progressive motility at 0 (both P < 0.0001) and 1 (P < 0.001; P < 0.01, respectively) h, and STR (both P < 0.05) and BCF (both P < 0.001) at 0 h. Sperm viability and acrosome integrity (both P < 0.001) were improved at both time points. Sperm frozen in Green Buffer had greater total and progressive motilities at 0 (both P < 0.001) and 1 (both P < 0.001) h than INRA-96® samples. Spermatozoa in Green Buffer also had a greater VAP, VCL and VSL at 0 h and improved viability and acrosome integrity at 0 h (P < 0.05; P = 0.001, respectively) and 1 h (P < 0.05; P < 0.001, respectively). Viability of SLC spermatozoa was improved in Green Buffer at 1 h (P < 0.05). Oocyte penetration (P < 0.05) and pronuclear formation (P < 0.01) were greater with SLC-selected spermatozoa than non-selected spermatozoa, regardless of extender. No difference was observed between treatments or extenders in the mean number of spermatozoa per oocyte penetrated. The SLC spermatozoa had less (P < 0.01) DNA fragmentation compared to controls. The DNA fragmentation was moderately and negatively correlated with penetration (r = −0.4162; P = 0.02) and pronuclear formation (r = −0.3390; P < 0.01). In conclusion, colloid centrifugation of spermatozoa and cryopreservation in Green Buffer improves post thaw motility variables and IVF performance of dromedary camel spermatozoa.

Differential influence of ampullary and isthmic derived epithelial cells on zona pellucida hardening and in vitro fertilization in ovine

The central role of the oviduct, as the site of zona pellucida (ZP) maturation, fertilization and early embryogenesis, has been recognized. The objective of this study was to investigate whether ampullary and isthmic derived epithelial cells have different effects on in vitro ZP hardening, in vitro fertilization (IVF) and in vitro culture (IVC) of the resulting embryos. Cumulus oocyte complexes (COCs) were matured in a coculture system with ampullary/isthmic epithelial cells, TCM199 supplemented with insulin-like growth factor I (IGF-I) and epithelial derived growth factor (EGF) (GF treated group), conditioned media produced using ampullary (ACM), isthmic (ICM), COCs+ampullary, and COCs+isthmic epithelial cells, contactless culture system, oviductal fluid, GF+ACM/ICM, and drops of TCM199 (control), for 24h. The matured oocytes were randomly divided into two groups: Group I was subjected to ZP digestion; Group II underwent IVF. The duration of the ZP digestion, in a coculture system with ampullary epithelial cells (AE) was significantly increased (p<0.05), compared with other groups. Penetrated oocytes and monospermic fertilization were significantly increased (p<0.05) in the AE group. The mean number of spermatozoa per penetrated oocyte was reduced dramatically for the AE group (p<0.05). A significant increase (p<0.05) in the embryo development was observed in all treated groups, compared to the control. Results revealed that epithelial cells harvested from the ampullary segment of the oviduct had in vitro specialized role in ZP hardening and have subsequent IVF and IVC outcomes.

Female major histocompatibility complex type affects male testosterone levels and sperm number in the horse (Equus caballus).

Odours of vertebrates often contain information about the major histocompatibility complex (MHC), and are used in kin recognition, mate choice or female investment in pregnancy. It is, however, still unclear whether MHC-linked signals can also affect male reproductive strategies. We used horses (Equus caballus) to study this question under experimental conditions. Twelve stallions were individually exposed either to an unfamiliar MHC-similar mare and then to an unfamiliar MHC-dissimilar mare, or vice versa. Each exposure lasted over a period of four weeks. Peripheral blood testosterone levels were determined weekly. Three ejaculates each were collected in the week after exposure to both mares (i.e. in the ninth week) to determine mean sperm number and sperm velocity. We found high testosterone levels when stallions were kept close to MHC-dissimilar mares and significantly lower ones when kept close to MHC-similar mares. Mean sperm number per ejaculate (but not sperm velocity) was positively correlated to mean testosterone levels and also affected by the order of presentation of mares: sperm numbers were higher if MHC-dissimilar mares were presented last than if MHC-similar mares were presented last. We conclude that MHC-linked signals influence testosterone secretion and semen characteristics, two indicators of male reproductive strategies.

Collection and characterization of semen from Chilean rose tarantulas (Grammostola rosea).

To establish a nonterminal semen collection method for use in captive Chilean rose tarantulas (Grammostola rosea) and to evaluate tools for investigating morphology and viability of spermatozoa. 7 mature male Chilean rose tarantulas. Each tarantula was anesthetized in a 500-mL induction chamber containing a cotton ball infused with 2 mL of isoflurane. Semen collection was performed by applying direct pressure to the palpal bulbs (sperm storage organs) located on the distal segment of the palpal limbs. Morphology of spermatozoa was examined by light microscopy and transmission and scanning electron microscopy. Propidium iodide and a fluorescent membrane-permeant nucleic acid dye were used to evaluate cell viability. Semen was collected successfully from all 7 tarantulas. Microscopic examination of semen samples revealed coenospermia (spherical capsules [mean ± SD diameter, 10.3 ± 1.6 μm] containing many nonmotile sperm cells [mean number of sperm cells/capsule, 18.5 ± 3.8]). Individual spermatozoa were characterized by a spiral-shaped cell body (mean length, 16.7 ± 1.4 μm; mean anterior diameter, 1.5 ± 0.14 μm). Each spermatozoon had no apparent flagellar structure. The fluorescent stains identified some viable sperm cells in the semen samples. The described technique allowed simple and repeatable collection of semen from Chilean rose tarantulas. Semen from this species was characterized by numerous spherical capsules containing many nonmotile spermatozoa in an apparently quiescent state. Fluorescent staining to distinguish live from dead spermatozoa appeared to be a useful tool for semen evaluation in this species.

Current knowledge on the transport and fate of spermatozoa in the reproductive tract of the bitch.

Canine sperm transport, distribution, storage and detachment is a complex, dynamic and highly regulated process. Transport of sperm within the bitch's reproductive tract is rapid and is influenced by the method of semen deposition (natural mating or artificial insemination) and by the timing of breeding in relation to the day of ovulation. The fertile lifespan of spermatozoa in the reproductive tract of the bitch is considerably longer than in most other domestic species, and the main sperm reservoirs appear to be the uterine crypts and the distal part of the uterotubal junction, where spermatozoa attach by their heads to uterine epithelium. While several in vitro studies demonstrated prolonged motility and viability of canine spermatozoa after coincubation with uterine tube explants, spermatozoal storage has not been documented in the canine uterine tube isthmus or ampulla in vivo. Several factors, including exposure to progesterone, solubilized zona pellucida proteins and post-ovulation uterine tube fluid, appear to trigger membrane events resulting in capacitation-like changes with subsequent motility pattern changes (transitional and hyperactivated) that are associated with sperm detachment. After mating or insemination, a normal low-magnitude post-mating uterine inflammatory response occurs, evidenced by an influx of polymorphonuclear neutrophils (PMNs), increased uterine contractions and an increased uterine artery blood flow. Recently, it was also shown that normal dogs with cystic endometrial hyperplasia develop a more significant endometritis, show fewer mating-induced uterine contractions, a decreased ability of spermatozoa to bind to uterine explants in vitro and a slower uterine clearance after mating.

PEMBERIAN VIRGIN COCONUT OIL UNTUK MENINGKATKAN JUMLAH DAN MOTILITAS SPERMATOZOA: STUDI PADA TIKUS WISTAR DENGAN DIET TINGGI LEMAK

Obesity is one of several causes of infertility in men, this accurs because off changes lifestyle, accurring in the diet. Consuming foods high in cholesterol and fatty foods can trigger obesity. High-fat diet can increase the production of free radicals that have a negative impact on the quality and amount of sperm motility. Virgin Coconut Oil contains lauric acid and polyphenols, which can capture free radicals. Objective of this study was to prove that Virgin Coconut Oil administration might increase the number and motility of spermatozoa in Wistar rats with high-fat diet (HFD). The study using Randomized Posttest Only Control Group Design. Thirty male Wistar rats aged 2 months and weighing 150-200 grams were divided into 5 groups: negative control (normal diet), positive control (HFD), first treatment group (HFD with VCO 1 ml/day), second treatment group (HFD with VCO 1.2 ml /day), and third treatment group (HFD with VCO 2 ml/day). The mean number of sperm in three treatment groups increase compared to the positive control group (375 million/ml), P1 (566.67 million/ml), P2 (510 million/ml) and P3 (500 million/ml), however statistically was went significant (p = 0.1). The mean percentage of sperm motility in the 3 treatment groups increase compared to the positive control group, P1 (43.33%), p2 (36%) and P3 (32.50%). however, statistically was not sinificant (p = 0.2). Virgin Coconut Oil cannot increase the number and motility of spermatozoa in male Wistar rats with hig-fat diet (p > 0.05). Keywords: high fat diet, virgin coconut oil, sperm number, sperm motility

The Effects of Frequent Electroejaculation on the Semen Characteristics of a Captive Siberian Tiger (&lt;i&gt;Panthera tigris altaica&lt;/i&gt;)

Artificial insemination (AI) can help to avoid inbreeding and genetic degeneration for sustaining genetically healthy populations of endangered species in captivity. Collection of a sufficient quantity of viable sperm is an essential first step in the AI process. In the present study, we examined the effects of frequent electroejaculation on semen characteristics in a Siberian tiger. We collected semen in all 17 trials during 6 breeding seasons (6 years). The mean number of sperm and the percentage of motile sperm were 294.3 ± 250.2×106/ejaculate and 82.4 ± 11.4%, respectively. The number of motile sperm tended to increase during frequent electroejaculation in the same breeding season. Semen collection by electroejaculation can be performed effectively up to the fourth sequential ejaculate, which contained the most sperm in the study. In conclusion, frequent collection of sperm by electroejaculation from tigers may be effective for collection of a large number of motile sperm.

227 CALRETICULIN, A 60-kDa PROTEIN, PREVENTS POLYSPERMY IN ZONA PELLUCIDA-FREE PIG OCYTES

After gamete membrane fusion or artificial oocyte activation, cortical granules undergo exocytosis and the released content modifies the zona pellucida (ZP), preventing polyspermy. Calreticulin (CRT), a calcium-binding highly conserved protein of 60 kDa, is contained in cortical granules from hamster eggs (Muñoz-Gotera et al. 2001 Mol. Reprod. Dev. 60), and we recently showed it is exocytosed from chemically activated ZP-free pig oocytes (Romar et al. 2012 Reprod. Fertil. Dev. 24). When pig ZP-enclosed oocytes were incubated with CRT, monospermy was not improved (Romar et al. 2011, Maternal communication with gametes and embryo, p. 72), suggesting that the likely role of CRT in preventing polyspermy might be carried out at the oolemma level. Our objective was to evaluate whether CRT prevents polyspermy in pig ZP-free oocytes by treating the cells with this protein before being inseminated. In vitro-matured cumulus–oocyte complexes (44 h, NCSU-37 medium) were decumulated and ZP was digested with Tyrode’s acid. The ZP-free oocytes were incubated for 30 min in TALP medium supplemented with 0, 100, 1000, and 5000 pg of CRT (ab91577, Abcam, Cambridge, MA, USA) per oocyte. After washing, ZP-free oocytes were inseminated (25 000 sperm mL–1) and gametes were co-cultured for 18 h. Putative zygotes were fixed and stained with Hoechst 33342 to analyse the fertilization results. Four replicates with 30 to 35 oocytes per group were done, and results were analysed by one-way ANOVA. A P-value ≤0.05 was taken to denote statistical significance. Incubation with CRT did not affect penetration rates that were similar among groups (77.12 ± 3.88 and 72.73 ± 4.07, respectively, for the 0- and 5000-pg CRT groups). However, the mean number of sperm per penetrated oocyte decreased from 3.01 ± 0.28 (0-pg group) to 2.07 ± 0.16 (5000-pg group), and monospermy rate increased from 30.77 ± 4.87 (0-pg group) to 52.27 ± 5.36 (5000-pg group; P ≤ 0.05). Incubation with CRT did not affect the number of sperm attached to oolemma, which was similar among all groups (11.45 ± 1.16 v. 10.75 ± 1.17, respectively, for 0 and 5000 pg of CRT). These preliminary data suggest that CRT, a protein exocytosed after oocyte activation, participates in the membrane block to polyspermy in pigs. Future studies to describe the exact mechanism of action of this chaperone protein are necessary. Supported by MEC and FEDER (AGL2009-12512-C02-01).

Distribution and viability of spermatozoa in the canine female genital tract during post-ovulatory oocyte maturation.

BackgroundUnlike other domestic mammals, in which metaphase-II oocytes are ovulated, canine ovulation is characterized by the release of primary oocytes, which may take 12 to up to 36 hours. Further 60 hours are needed for maturation to secondary oocytes which then remain fertile for about 48 hours. Oestrus takes 7 to 10 days on average and may start as early as a week before ovulation. This together with the prolonged process of post-ovulatory oocyte maturation requires an according longevity of spermatozoa in the female genital tract in order to provide a population of fertile sperm when oocytes have matured to fertilizability. Therefore the distribution and viability of spermatozoa in the bitch genital tract was examined during post-ovulatory oocyte maturation.MethodsThirteen beagle bitches were inseminated on the day of sonographically verified ovulation with pooled semen of two beagle dogs containing one billion progressively motile spermatozoa. Ovariohysterectomy was performed two days later (group 1, n = 6) and four days later (group 2, n = 7). The oviduct and uterine horn of one side were flushed separately and the flushing’s were checked for the presence of gametes. The oviducts including the utero-tubal junction and the uterine horns, both the flushed and unflushed, were histologically examined for sperm distribution.ResultsThe total number of spermatozoa recovered by flushing was low and evaluation of viability was limited. Prophase-I oocytes were collected from oviduct flushing in group 1, whereas unfertilized metaphase-II oocytes were detected in group 2. From day 2 to day 4 after ovulation a significant decrease in the percentage of glands containing sperm (P<0.05) and a marked reduction of the mean sperm number in uterine horn glands were observed. A concomitant diminution of spermatozoa was indicated in the utero-tubal junction accompanied by a slight increase in sperm numbers in the mid oviduct.ConclusionsOocyte maturation to metaphase-II stage is accompanied by a continuous sperm detachment and elimination in the uterine horns. Entrance of spermatozoa into the caudal oviduct seems to be steadily controlled by the utero-tubal junction thus providing a selected sperm population to be shifted towards the site of fertilization when oocyte maturation is completed.

Effect of dietary n-3 series fatty acids on sperm production in the freshwater crayfish, Astacus leptodactylus (Eschscholtz) (Astacidae)

The objective of the present work was to evaluate the effects of three different rates of long chain n-3 polyunsaturated fatty acid (LC n-3 PUFA) on the sperm number of Astacus leptodactylus. The experiment was carried out with the four following treatments: control group (not supplemented LC n-3 PUFA), D1 (1 %), D2 (2 %) and D3 (3 %). The sperm number of A. leptodactylus was counted; the reproductive system was weighted; and the macroscopical analysis of the testes and vasa deferentia (DVD) were conducted. In addition, gonado-somatic index (GI) and testes index (TI) were also calculated. The results showed that there was a significant difference between the crayfish fed LC n-3 PUFA supplemented diets (D1, D2, D3) and control in mean sperm number (P 0.05), but there was not a significant difference in the TI of the crayfish fed control, D1, D2 and D3 (P > 0.05). In conclusion, diets containing different levels of LC n-3 PUFA significantly increase the sperm number of A. leptodactylus, and they are crucial for the production of higher number of sperm in this species.

An investigation on the sperm number and reproductive parameters of males in wild caught freshwater crayfish (Astacus leptodactylus, Eschscholtz)

In the present study, the sperm number of wild caught Astacus leptodactylus was counted in the beginning of reproduction season. In addition, the reproductive system, testes, and vasa deferentia were weighed, and gonado-somatic index and testicular index were calculated. Twenty-five adult males of A. leptodactylus were used for the study. Significant linear regressions were found between body length (and weight) and sperm count, reproductive system weight, testes weight and vasa deferentia weight (). The mean sperm number of vas deferens for the size range of 41-56 mm carapace length crayfish was 5.72 ± 4.54 × 109 sperm/distal vas deferens (DVD) section. Sperm number ranged from 4 × 108 to 8.5 × 109 sperm/DVD section. The results revealed that an increase in body weight causes an increase in sperm number. For example, mean sperm number was 3.87 ± 2.87 × 109 for the weight range of 15-24 g crayfish, 5.01 ± 2.62 × 109 for the weight range of 25-35 g crayfish and 7.37 ± 2.87 × 109 for the weight range of 36-42 g crayfish. In conclusion, this study provides some data on the male reproduction characters and needs to be improved with details about the reproductive performance of males (i.e., sperm number and quality) in order to reach a maximum reproductive efficiency in captivity.

The effect of remating on sperm number in the spermatophores ofTeleogryllus commodus(Gryllidae)

Factors controlling sperm production in members of the Orthoptera have not been fully elucidated. In this study, the influence of intermating interval and ad libidum mating on sperm number was investigated in black field crickets (Teleogryllus commodus). Remating at a variety of time intervals was not characterized by a significant change in sperm number compared with the first mating. Ad libitum mating (i.e., continuous availability of unmated females) had two main effects on spermatophore production and sperm number: first, there was a trend toward increased time between copulations with each successive remating, and second, the number of spermatozoa encapsulated in the transferred spermatophore declined after most rematings, with 61.8% of the initial sperm number being produced for the second spermatophore and 51.3% of the initial sperm number being produced for the third. The decrease in mean sperm number was accompanied by increased variance in sperm number in later rematings. This study suggests that males are willing to suffer a decrease in sperm number if a mating opportunity occurs before the completion of sperm production.

Análise espermática de perdizes (Rhynchotus rufescens) criadas em cativeiro e suplementadas com selênio

Considerando a importância comercial da perdiz (Rhynchotus rufescens), implantaram-se algumas biotecnologias reprodutivas. Trinta animais, provenientes da FCAV-UNESP/Jaboticabal (2007-2008), foram aleatoriamente distribuídos em grupos: controle (sem selênio orgânico) e tratamento (com 0,2 a 0,8 mg de selênio em 1000 kg de ração ). Coletou-se sêmen por excitação manual, que foi aliquotado em pools com 150 µL. Mensuraram-se volume, motilidade, vigor, número de espermatozoides, concentração e morfologia espermáticas. Diluíram-se 20 µl de sêmen em 300 µl de solução fisiológica para testes complementares (Integridade das membranas acrossomal e plasmática). Contaram-se 200 células, por teste, e classificaram-nas: 1) Acrossomo Íntegro: cor lilás e Não-Íntegro: róseo; 2) Células Vivas (não coradas) e Mortas (coradas). Os dados foram analisados pelo SAS, System for Windows. Os resultados dos pools com e sem selênio foram: as variáveis volume, motilidade, vigor, número de espermatozoides, concentração, integridade acrossomal e integridade da membrana plasmática não apresentaram resultados significantes, porém encontrou-se uma menor porcentagem de espermatozoides com peça intermediária fortemente dobrada, nos animais tratados com selênio em relação aos não tratados (1,33 ± 0,53 vs. 3,78 ± 0,69, respectivamente; p = 0.0107). De fato, sabe-se que o selênio tem papel importante na estrutura dos espermatozoides20. A deficiência de selênio está associada com danos na arquitetura da peça intermediária do espermatozóide10.

Testicular morphology and antispermatogenic effects of 2,4-dichlorophenoxyacetic acid (2,4-D) in male West African Dwarf (WAD) goats

This study investigated the testicular morphology as well as the gonadal and extra-gonadal sperm reserves of West African Dwarf (WAD) goats exposed to graded levels of 2,4-dichlorophenoxyacetic acid (2,4-D). Twenty male WAD goats of five goats per group were used for this study. Goats in groups A, B and C received low [75 mg/kg body weight (BW)], medium (100 mg/kg BW) and high (125 mg/kg BW) dose levels of 2,4-D, respectively. The group D goats served as the control. On day 112, goats in the four groups were sacrificed and the testicular and epididymal sperm reserves were determined. Histopathologic changes in the testis of the 2,4-D-exposed and control goats were also assessed. The mean number of spermatozoa in the testes and the various segments of the epididymides decreased significantly (p < 0.05) in all the treatment groups relative to the control. Combined testicular sperm reserve per millilitre for the treatment groups (group A—19.61 ± 2.63 × 108, group B—12.02 ± 1.02 × 108 and group C—9.95 ± 0.97 × 108) reduced significantly (p < 0.05) relative to the mean value (23.52 ± 4.43 × 108) of the control—group D. The total epididymal sperm reserve per millilitre in the treatment groups (group A—24.25 ± 4.19 × 108, group B—17.18 ± 2.57 × 108 and group C—17.88 ± 2.89 × 108) was also found to be significantly (p < 0.05) lower than the mean value (40.85 ± 11.24 × 108) for the control—group D. This reduction in the testicular and epididymal sperm counts of the 2,4-D-exposed WAD goats in this study suggest disruption in spermatogenic activity, which may lead to low productivity. Variable degrees of circulatory disturbances were observed in the testis sections of 2,4-D-exposed goats.

Effects of centrifugation through three different discontinuous Percoll gradients on boar sperm function

In this study, different combinations of 2-step, discontinuous gradient centrifugation were used, consisting of three different combinations of isotonic Percoll (45/60, 60/75 and 45/90%) that allowed us to select different sperm subpopulations from fertile and normozoospermic boars. Our objective in this study is to evaluate the effects of centrifugation through three different discontinuous Percoll gradients on sperm function parameters (motility, viability, morphology, acrosome status, chromatin condensation, DNA fragmentation, ROS generation, tyrosine phosphorylation and intracellular calcium concentration) and the sperm penetrating capacity in an IVF system. All the Percoll treatments evaluated increased the percentage of spermatozoa with normal morphology, the proportion of un-damaged DNA, normal chromatin condensation, motion parameters measured by CASA and the percentage of capacitated spermatozoa with tyrosine phosphorylated proteins compared to control group. Finally, the in vitro oocyte penetrating capacity of boar spermatozoa was significantly affected by Percoll centrifugation. All the Percoll treatments increased the penetration rates and mean number of sperm per penetrated oocyte. Despite the efficiency of all three of the sperm treatments tested in selecting spermatozoa with improved sperm parameters and capacity to penetrate oocytes in vitro, the optimum performance of this system was demonstrated after preselecting spermatozoa by centrifugation on a discontinuous 45/90 Percoll gradient. The P45/90 treatment leads to obtain a higher percentage of spermatozoa which develop properly the capacitation process as it was shown measuring tyrosine phosphorylation and intracellular calcium concentration.