Antioxidant combinations are no more beneficial than individual components in combating ram sperm oxidative stress during storage at 5°C

The unfrozen storage of ram semen for 2-4 days is an important goal for the acceptance of artificial insemination in sheep breeding programmes. The objective was to investigate the benefits of antioxidant supplementation on the production of hydrogen peroxide (H(2)O(2)) by ram spermatozoa stored at 5°C over 3 days. Ejaculates from 9 rams were split between two defined diluents, INRA-96 and RSD-1, and cooled slowly to 5°C for storage. Four different additives (vitamin E phosphate 6-100 μmol/L, catalase 500 IU+superoxide dismutase 9-150 μmol/L, and glutathione peroxidase, 20 IU) were investigated both separately and in combination. The amount of H(2)O(2) generated was assessed by use of a 1-step fluorometric micro-plate assay. Sperm viability, acrosome integrity and membrane fluidity were assessed by flow cytometry. H(2)O(2) production in INRA-96- compared with RSD-1-diluted spermatozoa increased approximately 2-3-fold after 24h in storage at 5°C and then declined up to 72 h, while that in RSD-1 showed no change over 72 h; this had no effect on the sperm characteristics. Addition of antioxidants singly reduced H(2)O(2) production in INRA-96, regardless of concentration. Optimal concentrations were vitamin E phosphate 12.5 μmol/L, catalase 500 IU, superoxide dismutase 37 μmol/L, and glutathione peroxidase, 20 IU. In any combination, none was more effective than others. Viability was reduced but acrosomal integrity protected by the antioxidants in INRA-96 but not in RSD-1; membrane fluidity was not affected. Based on this study, there were no combinations more efficient at combating oxidative stress than any one alone.     

Effect of storage of domestic cat (Felis catus) epididymides at 5 °C on sperm quality and cryopreservation

Postmortem sperm recovery from the epididymides may constitute a powerful tool for the conservation of valuable genetic material. The domestic cat (Felis catus) is a good model for wild felids and, using this model, we have explored the effect of epididymides storage time on sperm motility and percentage of intact acrosomes upon sperm recovery and after cryopreservation. We also examined the effect of time of sperm equilibration with glycerol before freezing on sperm motility and the percentage of intact acrosomes. Motility varied between sperm recovered from epididymides that were stored for different times. Significant differences were seen in the sperm motility index (SMI) before freezing (55.91 ± 2.02, 48.21 ± 1.47, and 43.03 ± 1.32) and after thawing (51.81 ± 3.02, 41.90 ± 2.14, and 42.35 ± 1.95) of sperm recovered from epididymides stored for 0, 48, or 72 h, respectively. The percentage of intact acrosomes did not vary significantly with storage time (average 60.33 ± 1.38% before and 52.50 ± 1.91% after freezing, respectively). The percentage of normal sperm after different storage times did not differ (average 19.22 ± 1.25% normal sperm after recovery). When epididymides were stored for 72 h, time of sperm equilibration with glycerol (30 vs. 120 min) resulted in significant differences in both motility (SMI = 39.17 ± 2.76 and 45.00 ± 2.65, respectively) and the percentage of intact acrosomes (45.76 ± 4.91% and 60.67 ± 3.64%, respectively) after thawing. In conclusion, best results are achieved when sperm are recovered from epididymides within 24 h of cool storage and when they are equilibrated with glycerol during 120 min before freezing. The current results should be useful in the further development of techniques for the rescue and cryostorage of epididymal spermatozoa of endangered felids.     

The effects of different sugars on motility, morphology and DNA damage during the liquid storage of rat epididymal sperm at 4°C

This study evaluated the protective effects of supplementation with three different sugars on the motility, morphology and DNA integrity of rat epididymal sperm chilled and stored at 4°C Epididymides were obtained from each donor. Rat epididymal sperm was diluted in Ham's F10 plus raffinose, Ham's F10 plus trehalose, Ham's F10 plus fructose, and Ham's F10 medium for control purposes. Thereafter, the extended sperm were chilled and stored in liquid form at 4°C. Sperm motility, morphological abnormalities and DNA damage were determined at 0 and 12h after chilling. No significant difference was observed in any of the parameters evaluated at 0h, before storage (P>0.05). After 12h of storage, all sugar additives led to statistically higher motility, normal sperm morphology and DNA integrity in comparison to the control group. Raffinose gave the best motility percentages (32.86±1.84%) after 12h of storage at 4°C, compared to the other groups (P<0.001). In conclusion, Raffinose, trehalose and fructose provided a better protection of sperm functional parameters against chilling injury, in comparison to the control group.     

Effects of green tea polyphenol on the quality of canine semen after long-term storage at 5 °C

The aim of the present study was to determine the effects of green tea polyphenol on the quality of canine semen after long-term storage at 5 °C. The supplementation of a Tris-egg yolk extender with polyphenol (0.5, 0.75, or 1 mg/mL) increased the motility and viability of sperm preserved for four weeks at 5 °C.     

Ram seminal plasma improves pregnancy rates in ewes cervically inseminated with ram semen stored at 5 °C for 24 hours

In this study, we compared pregnancy rates obtained using ram semen stored at 5 °C for 24 h, with ram or bull seminal plasma (SP) added to TRIS-egg yolk extender. During the breeding period, 670 adult Corriedale ewes were cervically inseminated with semen (2 × 10⁸ sperm in a volume of 0.2 mL) from eight adult Corriedale rams. Ejaculates, obtained using an artificial vagina, were split into three aliquots and diluted with the following: TRIS-egg yolk based extender (T), T + 30% ram SP (R), or T + 30% bull SP (B). Samples were refrigerated and stored at 5 °C for 24 h until used for AI. Pregnancy was assessed by ultrasonography 35 to 40 d after AI. Pregnancy rate was not affected by ram (P = 0.77) or breeding period (P = 0.43), and there were no interactions between extender and ram (P = 0.94), or extender and breeding period (P = 0.24). However, there was an effect of extender (P = 0.0009) on pregnancy rates; ram SP, but not bull SP, increased pregnancy rates compared with extender without SP (49.7, 38.1, and 31.1%, for R, B, and T respectively). In conclusion, ram SP added to TRIS-egg yolk extender had a beneficial effect on the pregnancy rate of ram sperm stored at 5 °C for 24 h and used for cervical insemination of ewes.     

Short-term storage sperm of coho salmon (Oncorhynchus kisutch) at 4 °C: Effect of sperm: Extender dilution ratios and antioxidant butyl-hydroxytoluene (BHT) on sperm function

Short-term storage of semen is a necessary key procedure in fish; it allows maximizing the use of gametes. Nevertheless, sperm quality decreases during storage has been associated with oxidative stress damage due to an increase in reactive oxygen species (ROS) during storage. This study was designed to optimize a short-term storage protocol for Coho salmon (Oncorhynchus kisutch) spermatozoa, evaluating the effect of extender dilution and the addition of butyl-hydroxytoluene (BHT) antioxidant on sperm function parameters. In the first experiment, fresh semen was diluted in Storfish®: extender dilution (1:2 and 1:3) and a control sample undiluted and stored at 4 °C for 7-days. In both experiments motility (MO), viability and integrity of plasma membrane, mitochondrial membrane potential (MMP) and superoxide anion level (O₂⁻) were evaluated at 0, 3 and 7 days. Result shows that, 1:3 dilution maintained a higher sperm function for a longer period time. In the second experiment, spermatozoa were suspended in Storfish® (1:3) supplemented with two different concentrations of BHT (1.0 mM and 2.0 mM) and a control sample without antioxidant and stored at 4 °C for 7 days. The results demonstrated that, antioxidant-supplemented samples greater MO than control samples (P < 0.05). The viability remained >75% during storage in all groups. MMP was higher in 2.0 mM BHT compared to 1.0 mM and control (P < 0.05), in addition, this concentration reduced O₂⁻ level (P < 0.05). In conclusion, sperm: extender dilution 1:3 and adding of 2.0 mM BHT in sperm storage extender may enhance protection sperm function in Oncorhynchus kisutch against effects harmful of the oxidative stress during the in vitro storage.     

Changes in sperm membrane and ROS following cryopreservation of liquid boar semen stored at 15 °C

Boar semen is occasionally transferred to different locations in liquid form at 15 °C for cryopreservation. However, the use of frozen boar semen is limited due to the high susceptibility of boar sperm to cold shock. The aim of this study was to help improve the quality of frozen boar semen by determining the changes in sperm membrane and ROS during the cryopreservation processes of 15 °C-stored boar semen. Semen was collected from ten Duroc boars and transferred to our laboratory in liquid form stored at 15 °C. After cooling to 5 °C and freezing-thawing, conventional sperm parameters (total motility, progressive motility, and normal morphology), plasma membrane integrity, acrosomal membrane status, and intracellular ROS were evaluated. Sperm function, as assessed by conventional parameters, was unaffected by cooling but was decreased by freezing-thawing (P<0.05). However, the cooling and freezing-thawing processes led to damages in the sperm plasma membrane, and the cooling process caused increase in mean PNA (peanut agglutinin)-fluorescence intensity in viable acrosome-intact sperm (P<0.05). In ROS evaluation, the cooling process decreased intracellular (·)O(2) and H(2)O(2) in viable sperm (P<0.05), while the freezing-thawing process increased intracellular H(2)O(2) (P<0.05) without change in intracellular (·)O(2) in viable sperm. Our results suggest that, in liquid boar semen stored at 15 °C, cooling may be primarily responsible for the destabilization of sperm membranes in viable sperm, while freezing-thawing may induce reductions in sperm function with increase in membrane damage and H(2)O(2).     

Biosynthetic capacity of hepatocytes after storage at 4°C

Capacity to synthesize glucose, urea, and ketone bodies is well maintained in hepatocytes after storage for at least 24 h at 4 degrees C. Substrates and albumin are the only requirements.     

Fertility of undiluted ram epididymal spermatozoa stored for several days at 4°C

In vitro preservation of the male gamete is a challenge in the development of artificial insemination techniques for domestic animals. Specific strategies and diluents have been developed for the preservation of the fertilizing ability of the semen for each species. However, the epididymal medium has been demonstrated to be the best sperm environment to maintain sperm viability over several days and weeks for mammals. The aims of this study were to evaluate the motility and in vivo fertility of ram epididymal spermatozoa when the semen was stored for up to 4 days at 4°C undiluted in epididymal plasma. The study was undertaken with two ovine breeds (Ile de France and Corriedale). The motility of epididymal spermatozoa was better preserved in the undiluted epididymal fluid than when epididymal spermatozoa were diluted in classic ovine extender such as skim milk. During storage, the decrease in the percentage of motile sperm was lower if the epididymal spermatozoa were collected immediately after epididymal sampling than 24 h after castration or animal death. The fertility obtained after cryopreservation of the stored sperm and subsequent intrauterine insemination ranged from 55% to 24% following 24 to 96-h sperm storage. There was a linear regression relationship between fertility and the number of motile sperm inseminated for both breeds. These results show that it is possible to keep epididymal sperm motile and fertile for several days without dilution. Such a method of sperm preservation could be a final possibility for animals of high genetic value or for endangered species when the collection of semen before death of the animal is not possible.     

Epigenetic reprogramming of embryos derived from sperm frozen at −20°C

Cryopreservation of spermatozoa is a strategy that has been used to conserve the sperm of animal species and animal strains that are valuable for biomedical research. A simple method for preserving spermatozoa after application of intracytoplasmic sperm injection (ICSI) is much needed. It has been shown previously that spermatozoa frozen at 20°C can activate oocytes and support full-term embryo development. However, epigenetic reprogramming could be affected by the environment and by the in vitro manipulation of gametes. Here, we investigated embryo epigenetic reprogramming including DNA methylation and histone modification, in embryos derived from sperm preserved at 20°C without cryoprotectants. The results showed that although both fertilization and embryo developmental competence were decreased, the dynamic epigenetic reprogramming of embryos derived from frozen sperm was similar to the reprogramming of embryos derived from fresh sperm. The results reported in this study indicate that sperm frozen without cryoprotectant is epigenetically safe for ICSI.     

Artificial insemination with cryopreserved sperm from feline epididymides stored at 4 °C

Recovering and storing sperm from the epididymides of males of rare felidae is useful for preserving the species. The objective of the present study was to determine pregnancy rates following artificial insemination (AI) of frozen-thawed epididymal sperm, which were cryopreserved following low-temperature storage of the epididymides. In this study, these sperm were used for unilateral intrauterine AI (UIUAI) or unilateral intratubal AI (UITAI) using 40 × 10⁶ and 10 × 10⁶ sperm, respectively. The caudal epididymides of 17 cats were stored at 4 °C for 24 h after castration. Artificial insemination of seven female cats was performed on Days 3 or 4 (start of estrus = Day 1) by UIUAI, 20 h after injection of 100 IU hCG to induce ovulation. Furthermore, UITAI at 24 h (UITAI-24) or 30 h (UITAI-30) after hCG were also done (five cats per group). It was noteworthy that AI by UIUAI and UITAI-24 was performed before ovulation, whereas AI by UITAI-30 was performed after ovulation. Pregnancy rates were 28.6% (2/7) by UIUAI, 80% (4/5) by UITAI-24, and 20% (1/5) by UITAI-30. Litter size was one or two by UIUAI, and one to four by UITAI. Spontaneous abortion occurred on Days 25-30 of pregnancy in one of the two female cats pregnant following UIUAI, and in two of five female cats pregnant following UITAI. Based on the high pregnancy rate obtained with 10 × 10⁶ sperm in the UITAI-24 group (AI performed before ovulation), we concluded that this was the most appropriate method for AI with frozen-thawed epididymal sperm after initial low-temperature storage of epididymides.     

The effect of relaxin supplementation of in vitro maturation medium on the development of cat oocytes obtained from ovaries stored at 4 °C

Relaxin is a member of the insulin-like family of hormones that promotes growth in a number of reproductive tissues, including the granulosa and theca cells. Cat oocytes collected from cold-stored ovaries remain capable of maturing in vitro, but the developmental ability of the oocytes decreases after 24 h of cold storage. To improve the developmental ability of cat oocytes from cold-stored ovaries, we investigated the effect of relaxin supplementation of maturation medium on their meiotic ability and subsequent development. Cat oocytes were collected from ovaries stored at 4 °C for one day and cultured in maturation medium supplemented with different concentrations (0, 10, 20, and 40 ng/ml) of relaxin for 24 h. They were then fertilized in vitro for 12 h with frozen-thawed spermatozoa. After in vitro fertilization, the zygotes were cultured in synthetic oviduct fluid medium for 8 days. There were no significant differences in the maturation rates and glutathione contents of oocytes among the groups, irrespective of relaxin supplementation. The rate of blastocyst formation from oocytes matured with 10 ng/ml relaxin (16.0%) was higher (p < 0.05) than that from oocytes matured without relaxin (5.9%). Our findings indicate that supplementation of 10 ng/ml relaxin into maturation medium may improve blastocyst formation of cat oocytes after in vitro fertilization.     

Variability of physiological parameters of unacclimatized males during a two—hour cold stress of 5°C

The metabolic and temperature responses of 11 male Caucasians to a 2-hr exposure to 5 ± 1°C, 70 ± 2% RH were compared with control data obtained in an ambient environment of 28 ± 1°C, 45 ± 2% RH. The heat production increased during the cold exposure attaining an approximately stable level during the final 30 min. The group variability in response to the cold was greatest during the first 30 min and declined for the remainder of the cold exposure. All skin temperatures approached a stable value during the final 30 min of cold exposure. The correlation between mean skin temperature and thigh temperature was significant (p < 0.001) and the use of thigh temperature as an approximate mean skin temperature was suggested. The calculation of tissue conductance with or without the inclusion of heat exchanges due to changes in body heat content and respiratory losses was in agreement only during the final 30 min of cold exposure, thus indicating a stage of physiological equilibrium. All measured parameters except the toe and finger temperatures approached minimum variability of response during the final 30 min of cold exposure.

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Zusammenfassung Die Reaktionen des Stoffwechsels und der Hauttemperatur von 11 männlichen Angehörigen der weissen Rasse während einer 2-stündigen Exponierung bei 5 ± 1°C, 50–70 % RF wurden mit den Kontrollwerten bei 28 ± 1°C und 45 ± 2% RF verglichen. Während der Kälteexponierung stieg die Wärmebildung an und erreichte wie die Hauttemperatur in den letzten 30 Min ein ungefähr konstantes Niveau. Die Gruppenvariabilität war in den ersten 30 Min am grössten und liess dann nach. Die Korrelation zwischen mittlerer Hauttemperatur und Oberschenkeltemperatur war hochsignifikant (p < 0,001). Es wird vorgeschlagen letztere als mittlere Hauttemperatur zu verwenden. Die Berechnung der Gewebeleitfähigkeit mit oder ohne Einbeziehung des Wärmeaustausches als Folge von Änderungen des Wärmegehaltes des Körpers und Wärmeverlustes bei der Atmung stimmte nur während der letzten 30 Min. Alle gemessenen Parameter ausser der Zehen- und Fingertemperatur näherten sich während der letzten 30 Min der Kälteexponierung einer minimalen Variabilität. Dies weist auf ein physiologisches Gleichgewicht hin.

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Resume On a comparé le métabolisme et la température cutanée de 11 personnes de la race blanche caucasienne exposées durant 2 heures à une température de 5 ± 1°C et à une humidité relative de 70 ± 2% aux mêmes valeurs obtenues par 28 ± 1°C et 45 ± 2%. La production de chaleur a augmenté durant l'exposition au froid pour atteindre un niveau relativement stable durant les 30 dernières minutes. La variabilité du groupe quant à la réaction au froid fut très importante durant les 30 premières minutes. Elle a notablement diminué le reste du temps. Toutes les températures cutanées se sont stabilisées durant les 30 dernières minutes de l'exposition au froid. La corrélation entre la température de la peau et celle de la cuisse fut hautement significative (p < 0,001) et l'on propose d'utiliser cette dernière température comme température cutanée moyenne. Le calcul de la conductibilité des tissus en y incluant ou excluant les échanges de chaleur dus aux variations thermiques du corps ou les pertes imputables à la respiration n'est exact que pour les 30 dernières minutes. Tous les paramètres mesurés, à l'exception des températures des doigts et des orteils tendent vers un minimum de variabilité durant ce même laps de temps. Ceci indique qu'un état d'équilibre physiologique est alors atteint.

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Supported in part by United States Public Health Service Grant No. HD-00235; and the Air Force Office of Scientific Research, Office of Aerospace Research, United States Air Force, under AFOSR Grant No. 69-1653. Data analysis (on IBM System 360 Model 75 computer) was made possible by funds from the Special Research Resources Branch, Division of Research Facilities and Resources, National Institutes of Health.     

Transfer and subsequent growth and metabolism of Lactobacillus plantarum in orange juice medium during storage at 4 and 30°C

Aim: To investigate the physicochemical changes produced from growth and metabolism of Lactobacillus plantarum N4 in orange juice medium stored at 4 and 30°C after transferring from artificially inoculated oranges peal during extraction. Methods and Results: Lower than 2·0% of total of the N4 strain was recovered in juice extracted from inoculated oranges (about of 10⁹ CFU ml^?1) under assayed conditions. After that, the N4 strain grew 2·43 ± 0·09 log cycles in 48 h at 30°C. Sugars such as glucose and fructose and l ‐malic and citric acids were utilized, although at different rates and extent, yielding significant lactate and acetate amounts with a concomitant pH reduction. Ethanol, diacetyl, acetoin or 2,3 butilenglicol were undetected. During juice storage at 4°C bacterial counts, sugars composition and pH remained significantly unchanged as well as its sensory attributes. Conclusion: The transfer rate of L. plantarum N4 to freshly squeezed juice under adequate hygienic condition was low. At 30°C, the micro‐organism rapidly initiated growth, producing acids but not butter flavour compounds neither ethanol. Significance and Impact of the Study: The ability of this strain to survive in refrigerated juice without cause spoilage warrants further investigation to explore its potential use for biotechnology applications.