Ultrastructural changes in Marsupenaeus japonicus spermatozoa in the journey from the testis to the female thelycum

Marsupenaeus japonicus is one of the most important penaeid shrimp farmed in China. As a typical closed thelycum penaeid shrimp, the morphological changes in the spermatozoa during the journey from the testis to the female thelycum remain unclear in this species. Herein, an ultrastructural comparison of the spermatozoa in the testis (TE), vas deferens (VD), and the spermatophore in the male terminal ampoule (SA) and the female seminal receptacle (SR) of M. japonicus was conducted. The spermatozoa in the VD, SA and SR shared typical morphological characteristics of the spermatozoa of decapod dendrobranchiates, including a spherical main body and a slender spike. However, no spike was observed in the spermatozoa collected from the TE. The spike of the spermatozoa in the SA and SR was longer than that in the VD, whereas no significant difference in spike length was observed between the spermatozoa from the SA and SR. The basic ultrastructure of the spermatozoa in the VD, SA and SR was similar, except for some differences in the nuclear region. The main body of the spermatozoa comprised the cytoplasmic band, nuclear region, several small and large vesicles, and the basal region of the acrosomal structure. The nuclear materials in the spermatozoa from the TE were more electron‐dense than those in the VD, SA and SR, while the nuclear region of the spermatozoa was more electron‐lucent in the SR than in the VD and SA. These findings preliminarily reveal the ultrastructural changes in M. japonicus spermatozoa during the journey from the testis to the female thelycum and provided a theoretical foundation for developing effective artificial insemination methods in this species.     

A fructose‐based extender protects Colossoma macropomum spermatozoa against chilling injuries

Our study assessed the efficiency of a formulated new extender in maintaining viability and morphological integrity of Colossoma macropomum spermatozoa under chilling storage. Semen was diluted in the test extender and BTS? (Beltsville Thawing Solution) and exposed to a short‐term storage at 4.6 ± 0.6°C for 96 hr. Both extenders were able to maintain 17% ± 8% motile spermatozoa by the end of experiment. Sperm dilution in test extender did not affect the morphologically normal cells (61% ± 6%) up to 48 hr of chilling, being higher than in BTS? (50% ± 6%) (p < 0.05). After 96 hr, samples kept in the test extender had 50% of normal spermatozoa, whereas those kept in BTS? presented only 38% of normal cells. Chilling storage increased the incidence of cells with strongly coiled flagella in BTS^?. Our study is the first to evaluate in detail the spermatozoa morphology as indicative of C. macropomum semen viability. The new extender was able to protect the spermatozoa against increase in coiled flagellum injuries.     

荧光染色法检测虹鳟精子质量

本实验运用三种荧光染色方法对虹鳟精子的质量进行检测,并探讨荧光染色对虹鳟精子质量评价的可性及意义。在生殖季节采集虹鳟精液,分别进行Hoechst33342/碘化丙啶双染,吖啶橙染色及罗丹明123/碘化丙啶双染,然后将其制成涂片荧光显微镜下观察。经荧光染色的虹鳟精子质膜完整,DNA完整,可明显分辨出活体和死亡精子,线粒体膜功能正常精子的比率分别为90.7%、98.7%和96.4%。综上所述,结合三种检测方法可以简便快速地测定虹鳟精子的质量。     

ATP content and sperm motility of extended bovine semen under different storage conditions.

The effects of incubation temperature (+20 degrees C vs +35 degrees C) and media type on the ATP content and motility of spermatozoa were determined in fresh bovine semen in order to develop a method for assaying post-thaw quality. Semen was obtained from 3 bulls at 2 occasions. The spermatozoa were washed using a Ficoll-containing medium before being resuspended in each of 4 different media (I. 0.9% NaCl; II. Tris-buffer solution; III. seminal plasma; IV. seminal plasma+Tris-buffer solution) and incubated for 6 h. The least-squares means for ATP content were higher (p less than or equal to 0.05) at +20 degrees C than +35 degrees C for all media except no. I. By contrast, the least-squares means for sperm motility were higher (p less than or equal to 0.05) at +35 degrees C than at +20 degrees C in media II and III. A decrease over time in ATP content and motility at both temperatures was also observed. The single most important factor responsible for changes in ATP content and sperm motility was the temperature and the medium, respectively.     

Membrane Alterations in Bull Spermatozoa after Freezing and Thawing and after In Vitro Fertilization

Membrane alterations in bull spermatozoa after freezing and thawing and after the process of in vitro capacitation and fertilization were studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Even if the majority of the spermatozoa exhibited intact membranes after freezing and thawing (90%), one could distinguish between 3 types of membrane defects depending of the different structures involved. The first type showed loss of plasmalemma over the entire acrosome. In the second category the anterior part of the outer acrosomal membrane exhibited a pronounced extension, but was covered by a partly intact plasmalemma. The last category consisted of spermatozoa with extensive vesiculation and disruption of plasmalemma and the outer acrosomal membrane. This type of defect could not easily be distinguished from a true acrosome reaction. The cumulus cells showed an active phagocytosis of both intact and acrosome reacted spermatozoa.     

间情期犬间母细胞体外受精的影响因素

试验对间情期犬卵母细胞体外受精的影响因素进行了系统研究。结果表明①以TALP液作为犬精子获能液，精子回收率(64±10.5)%、顶体反应率(41.5±4.1)%高于TCM199+20%FCS液。②用TCM199+20%FCS作为受精液，卵母细胞单精受精率(18.6%)、原核形成率(10.4%)，高于TALP受精液，而退化率低于TALP受精液(4.9%比19.7%，P＜0.01)。③犬精子与卵母细胞在受精液中共同孵育12h，单精受精率(23.5%)、原核形成率(12.7%)高于共同孵育8、16、20h，而多精受精率(15.7%)、退化率(3.9%)低于共同孵育16、20h。④犬精子为2×106个/mL时，单精受精率(26.0%)、原核形成率(14.6%)，高于0.5×106、1×106、3×106、5×106个/mL组。卵母细胞多精受精率和退化率随精子含量增加而增加。⑤肝素为29mg/L时,卵母细胞单精受精率(24.4%)、原核形成率(12.2%)高于0、10、20、40、50mg/L组。而犬精子获能2h后，顶体反应率和卵母细胞多精受精率随肝素质量浓度增加而增加。     

公牛精子分离前的保存

试验采用荷斯坦公牛精液，比较了添加抗生素、TALP稀释以及TALP稀释染色3种处理方法对常温下保存精子活力的影响。研究结果表明，新鲜精液加抗生素处理后直接保存的效果优于另外2个处理组，而分离精子经冷冻解冻后的存活率达40％，说明了该方法的可靠性。     

Sperm winding in Collembola

The winding process leading to the formation of rolled-up spermatozoa in Collembola is described. It is a phenomenon common to all collembolan species examined so far and it starts with the organization of a cytoplasmic flattened evagination beneath the head of the elongated spermatid, which gives rise to an extracellular cavity. Within this cavity secretory material produced by the epithelial cells of testes is progressively stored. The spermatozoon, whose cytoplasm is reduced to a thin layer, enrolls around this material. The final appearance is that of a spheroidal or lenticular structure containing dense material in the central extracellular cavity; this cavity is surrounded by the flattened sperm in whose cytoplasm the sperm components forms several spires. The material into the extracellular cavity becomes progressively denser and has a different appearance in the species examined.     

First successful frozen semen of the maned wolf (Chrysocyon brachyurus)

This study aimed to describe successful cryopreservation of sperm from maned wolves (Chrysocyon brachyurus). Three ejaculates from 2 maned wolves were collected by digital manipulation of the penis and evaluated subjectively, centrifuged and frozen in BotuCrio^® (Botupharma, Botucatu, Brazil) or Tris–yolk egg extender. Spermatozoa were thawed at 37ºC/30s or 70ºC/4s and evaluated for kinetics, morphology, plasma and acrosome membrane integrity, mitochondrial potential, hydrogen peroxide, superoxide anion and lipid peroxidation. From 5 thawed samples, two had sperm total motility >55% (56.0% and 64.0%) and progressive motility ~35% (35% and 40%), both frozen with Tris–yolk egg. Plasma and acrosome membrane integrity decreased and percentage of sperm defects increased post-thawing. We concluded that is possible to freeze spermatozoa from maned wolves using semen collection and processing methods applied for domestic dogs.     

New protocol to separate llama sperm without enzymatic treatment using Androcoll-E™

The objective of this study was to design a protocol to separate spermatozoa from seminal plasma of raw llama semen without prior enzymatic treatment using a single-layer centrifugation with Androcoll-E^™ (AE). Two experiments were performed: (a) samples were divided into three aliquots (1 ml) that were deposited on the top of 4, 5 or 6 ml of AE and were centrifuged at 800g for 20 min and (b) samples were divided into two aliquots (1 ml) that were deposited on the top of 4 ml of AE and were centrifuged at 600g or 1,000g for 20 min. Columns of 5 and 6 ml of AE showed a total sperm motility (TM) significantly lower, while in the 4 ml column, this parameter was not different from the TM of samples before the AE treatment. The percentage of spermatozoa with intact and functional membranes, normal morphology and intact acrosomes, as well as the percentages of sperm with highly condensed chromatin, was conserved (p ˃ .05) in the three column heights and in the two centrifugation speeds evaluated. In conclusion, the different column heights of AE (4, 5 and 6 ml) and the different centrifugation speeds used (600, 800 and 1,000g) allow separating spermatozoa of raw llama semen without enzymatic treatment, preserving the evaluated sperm characteristics. However, of all the studied treatments, centrifugation in the 4 ml column of AE at 800g would be the method of choice to process raw llama semen samples destined for reproductive biotechnologies.