Comparison of different osmolalities and egg-yolk composition in processing media for the cryopreservation of red wolf (Canis rufus) sperm

Successful cryopreservation of sperm and the maintenance of a sperm-based genome resource bank have been identified as priorities for the recovery of the endangered red wolf (Canis rufus). The objectives were to improve sperm processing and to determine the relative timing of damage to red wolf sperm during freezing and thawing. Fresh ejaculates (n=37) from adult red wolves (n=15, aged 2-13 y) were collected via electroejaculation and subjected to cooling, freezing and thawing in four TRIS-egg-yolk extender treatments varying in osmolality ( approximately 305 mOsm versus approximately 350 mOsm) and egg-yolk composition (0.8 microm-filtered versus unfiltered). Ejaculates were evaluated for sperm percentage motility, forward progressive motion, and morphological characteristics immediately upon collection and following extension, cooling (prior to freezing) and thawing. Although no single treatment consistently produced superior results, sperm suspended in approximately 305 mOsm extenders exhibited slight losses in motility post-thawing (13 and 7%). Also, sperm suspended in approximately 350 mOsm extenders tended to have slower rates of decline in motility in vitro post-thawing than those stored in approximately 305 mOsm extenders (P=0.55). Finally, extenders incorporating unfiltered egg yolk exhibited a slightly larger ratio of absent to partial acrosomes than did sperm frozen in extenders prepared with clarified egg yolk. For approximately 350 mOsm extenders, most motility loss occurred during the cooling rather than freezing and thawing. In conclusion, these data contribute to knowledge regarding cryopreservation of red wolf sperm.     

Freezability prediction of boar ejaculates assessed by functional sperm parameters and sperm proteins

The objective of this work was to look for useful predictive indicators of the potentially “good” or “poor” ability of a boar ejaculate to sustain cryopreservation by assessing both the conventional sperm quality parameters (Study 1) and the immunolabeling of three proteins involved in the physiology of the sperm cell: GLUT3, HSP90AA1 and Cu/ZnSOD (Study 2). Study 1 was carried out in three different steps during the cryopreservation process of the sperm-rich fraction of 29 Piétrain boar ejaculates (17 °C, 5 °C, and 240 min postthaw). These ejaculates were clustered based on sperm quality parameters analyzed at 240 min postthaw, obtaining 16 good freezability ejaculates (GFEs) and 13 poor freezability ejaculates (PFEs). The sperm linearity (LIN) and the straightforward (STR) indexes at 5 °C showed higher hyperactivated movement in the PFEs than in the GFEs, which suggests that analyzing these sperm kinematic parameters could be a useful tool for predicting the potential freezability of an ejaculate. This statement was demonstrated by grouping the 29 ejaculates into two clusters (A and B) based on LIN and STR values assessed after 30 min at 5 °C, which resulted in around 72% of coincidence with the GFE and PFE groups. Study 2, performed at 17 °C and 240 min postthaw, revealed no differences between GFEs and PFEs in the immunolabeling of the three proteins within a same step, in terms of location and reactivity, although reactivity was generally weaker at 240 min postthaw in both groups. Additional studies on Western blot are currently being carried out with the objective to quantify the expression of the three proteins in GFEs and PFEs in the three steps of the cryopreservation process.     

Effects of season and feeding level on reproductive activity and semen quality in Payoya buck goats

The aim of this study was to determine if there is a seasonal pattern of reproductive activity in male Payoya goats and if this seasonality can be modulated by a higher level of nutrition. For a period of 16 months, 10 adult bucks were divided into two experimental groups that differed in their feeding level. The high nutrition group (H, n = 5) received 1.6 times their maintenance food requirements. The control nutrition group (C, n = 5) received a diet that supported 1.1 times their maintenance requirements. Body weight and testosterone concentrations were determined weekly, and testicular weight was determined every 2 weeks. Sexual behaviour and semen characteristics were determined monthly. Feeding level did not affect the onset or the end of the reproductive activity as measured by testosterone concentrations, with high testosterone concentrations between July and November. Ejaculation latency was positively influenced by feeding level: 43.2 ± 2.2 s vs. 61.6 ± 3.2 s for H and C group, respectively (P < 0.001). The percentage of males that ejaculated or that were sexually active was higher in the H group (P < 0.01). No differences between feeding levels were observed in the different semen characteristics studied. However, major differences between months were observed for all studied variables. These results lead us to conclude that Payoya bucks exhibit large seasonal variation in their reproductive activity. Higher feeding level allowed a better sexual behaviour in bucks in late spring, when male effect is used on the local livestock to breed females.     

Male hypofertility induced by Paraquat consumption in the non-target parasitoid Anisopteromalus calandrae (Hymenoptera: Pteromalidae)

The effects of a herbicide – Paraquat – on male development and reproduction were tested on the parasitoid wasp Anisopteromalus calandrae (Hymenoptera, Pteromalidae) by injecting host larvae with different concentrations of this substance. Data measured were: (1) developmental success, (2) sperm stock in seminal vesicles, (3) ability to copulate and transfer sperm, and (4) offspring production. Both developmental success and sperm in seminal vesicles were reduced in the “Paraquat” groups. However, neither sperm stored in the females’ spermatheca, nor offspring production (number of female offspring and sex ratio) differed from controls, whatever the host treatment. The decreased of male sperm reserve in ‘‘Paraquat” group is likely to reduce their reproductive success because they can succefully inseminated less female than control males. These males are thus hyporfertiles. Because Paraquat has non-negligible consequences on non-target parasitoid males, it is likely to affect natural and controlled insect populations.     

Human sperm cryopreservation in cancer patients: Links with deprivation and mortality

Evidence is mounting for a relationship between human semen quality and environmental/lifestyle/socioeconomic factors including long term health outcomes such as mortality. The relationship between pre-freeze and post-thaw semen quality in cancer patients and these factors are unknown. Frozen semen from 217 cancer patients was thawed and analysed using a validated CASA method. Post-thaw quality was matched and compared with WHO semen analysis performed prior to storage. The English Indices of Deprivation 2010 were matched with patients and then examined for relationships with pre-freeze and post-thaw semen quality. There is a relationship between semen quality and deprivation in cancer patients. Compared with pre-freeze semen quality, post-thaw semen quality has a stronger relationship with deprivation. Sperm cryopreservation may have potential as a systemic health diagnostic test and is predictive of cancer patient mortality.     

How is a giant sperm ejaculator formed? Development of the Zenker organ after the last moult in Pseudocandona marchica (Crustacea,Ostracoda, Candonidae)

‘Giant sperm’, in terms of exceptionally long spermatozoa, occur in various taxa in the animal kingdom, particularly in arthropods. In freshwater ostracods (Cypridoidea) reproducing with giant sperm, males are equipped with a pair of specialized ejaculators, or Zenker organs, located in the distal part of vas deferens.     

The influence of recombinant feline oviductin on different aspects of domestic cat (Felis catus) IVF and embryo quality

Oviductin is known to be a key player providing a convenient environment for the process of fertilization affecting this by direct interaction with oocytes and sperm. As in vitro embryo production in the context of assisted reproduction for endangered felids is still in the process of optimization, oviductin might be used to improve IVF results. Recombinant His-tagged feline oviductin was expressed by transformed Escherichia coli BL21DE3 cells. The protein was purified by immobilized metal ion affinity chromatography. The effect of the recombinant protein was characterized in three experiments: a hemizona assay for sperm binding analysis, the IVF outcome, and the relative mRNA expression levels in blastocysts after IVF. A significant higher number of bound sperm cells were found after incubation in oviductin. No significant effect on cleavage, morula, and blastocyst rates with or without oviductin incubation during IVF could be observed. However, the relative mRNA abundance of GJA1, a gene, whose expression level is known to be a marker of embryo quality, was significantly increased (P value less than 0.05) in blastocysts after oviductin treatment. In contrast to this, expression of OCT4, HSP70, DNMT1, DNMT3A, BAX, IGF1R, and GAPDH was not significantly affected. We assume that our recombinant oviductin in its current nonglycosylated form is able to enhance sperm binding. Despite of a missing significant effect on IVF outcome, embryo quality in terms of relative GJA1 expression is influenced positively. These promising results demonstrate the value of recombinant oviductin for the IVF in cats.     

Study of nuclear and acrosomal sperm morphometry in ram using a computer-assisted sperm morphometry analysis fluorescence (CASMA-F) method

The aim of this study was to develop a new method that allows morphometric assessment of the sperm nucleus and acrosome in the ram using fluorescence microscopy and free software. The study was divided into three experiments. In the first experiment, semen smears from 20 ejaculates were fixed and labeled with a propidium iodide–pisum sativum agglutinin (PI/PSA) combination. Digital images of the sperm nucleus, acrosome, and whole sperm head were captured and analyzed using the ImageJ program. The computer-assisted sperm morphometry analysis fluorescence (CASMA-F) method used allowed the differentiation, capture, and morphometric analysis of most sperm nuclei, acrosomes, and whole heads with high precision and the assessment of the acrosomal status. In the second experiment, sperm nuclear morphometry by CASMA-F was compared by staining with the PI/PSA combination and staining with Hoechst 33342 as in previous studies. Similar results were obtained using both methods. In the third experiment, CASMA-F with PI/PSA was compared with a more conventional CASMA method (semen smears stained with Hemacolor (HEM) and processed with the ISAS commercial software, HEM). Spermatozoa displayed a bigger size when processed with CASMA-F than with HEM method in all primary sperm head morphometric parameters, but results using both methods were correlated. It was concluded that the CASMA-F method allows the simultaneous assessment of sperm nucleus, acrosome, and head in the ram.     

Cryopreservation of sperm from Atlantic halibut (Hippoglossus hippoglossus, L.) for commercial application

Development of Atlantic halibut (Hippoglossus hippoglossus) aquaculture will be enhanced with cryopreservation of halibut sperm by ensuring a reliable supply of sperm of desired quality and quantity. To assist in its commercial application, the cryopreservation of large volumes of halibut sperm was investigated. Three cryoprotectants were compared: dimethyl sulfoxide (DMSO), polyethylene glycol (PG) and glycerol (GLY) at two concentrations (10% or 15%). Two salt solutions, Hanks’ balanced salt solution (HBSS) and 0.1 M KHCO₃ with 0.125 M sucrose solution (KS) were tested as diluents. Both factors were examined in 1.6 mL volumes. A cryopreservation volume of 4 mL and a low dilution ratio (1:1) were examined separately. Based on motility and fertilization rate, 10% and 15% DMSO diluted with HBSS or KS solution proved to be effective extenders with mean fertilization rates ranging from 52.2 ± 27.2% to 65.8 ± 26.1%; none of which were significantly different from that of the control. Four other extenders, 10% PG or 10% GLY with HBSS or KS, resulted in significantly lower fertilization rates. Use of a 4 mL cryopreservation volume did not exhibit a significant effect on fertilization rate or motility of post-thawed sperm compared to a 1.6 mL volume (P > 0.05); while the use of a dilution ratio of one part sperm with three parts cryopreservation solution (1:3 v/v with sperm concentration of 0.51 ± 0.11 × 10¹⁰ cells/ml) had a significantly better preservation effect than using a ratio of 1:1 with sperm concentration of 1.02 ± 0.21 × 10¹⁰ cells/ml (P < 0.05). From these results, an optimized protocol for the cryopreservation of Atlantic halibut sperm using a volume as large as 4 mL has been established.