Storage Of Semen Research Articles

P-438 Fertility outcomes in male cancer recipients of hematopoietic cell transplantation who stored their semen for fertility preservation

Abstract Study question What are the prevalence estimates of post-hematopoietic cell transplantation (HCT) natural conceptions or after ART using stored semen? Summary answer Natural live birth rate following HCT can be estimated at 9% (95%CI:4-22%) but can be increased to 58% (95%CI:43-72%) with the use of frozen semen. What is known already With the improvements and expanding indications of HCT, the number of adult male cancer survivors who received HCT is increasing. These men face the risk of treatment-induced infertility and are generally recommended to store their semen prior to embark in HCT. However, information on long term usage and on effectiveness of frozen sperm samples in this specific population is not well defined. Study design, size, duration All male cancer recipients of autologous or allogeneic transplantation who cryopreserved semen between 1987 and 2016 in the biobank of the Assisted Reproductive Technology Unit of the Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico in Milan were considered. Information was obtained from patients’ charts or through active investigation. The main studied topics included results from semen analyses, attempt to parenthood, use of stored spermatozoa and both natural and Assisted Reproductive Technique (ART) mediated conceptions. Participants/materials, setting, methods The study is restricted to men who met the following eligibility criteria: i) semen cryopreserved for fertility preservation between 1987 and 2016; ii) post-banking follow-up documenting HCT iii) survival of at least 5 years from HCT. Main results and the role of chance One-hundred and two HCT survivors were selected. The median [IQR] age at sperm banking was 29 [23-34]. About half of subjects had autologous HCT (n = 50), the remaining 52 received allogenic HCT. Lymphomas were the most frequent indications. The median spermatozoa concentration at the time storage was 39 [7-85] millions/ml. Fifty-four (53%) men performed a post-treatment sperm analysis and azoospermia was documented in 45 of them (83%, 95%CI: 71-91%). The remaining nine showed viable spermatozoa, of whom six had severe oligospermia (concentration <1 million/mL). The other three had semen concentration >5 Millions/ml, compatible with a natural conception. Overall, based on semen analyses, one could infer the necessity to use ART in 51 out of 54 subjects, corresponding to 94% (95%CI: 85-98%). Forty-three of the 102 included men (42%) sought childbearing. Four had a natural live birth (9%, 95%CI: 4-22%). Thirty-three used their cryopreserved sperm samples, corresponding to 32% (95%CI: 24-42%) of the whole cohort. Twenty-one of these 33 men had at least one live birth (64%, 95%CI: 47-78%). Finally, six men seeking pregnancy and who did not conceive have not used their frozen semen. Overall, 25 out of 43 men interested in fatherhood had a live birth (58%, 95%CI: 43-72%). Limitations, reasons for caution Limitation of our study is missing detailed information on status of illness and comorbidities (in particular the frequency of Graft-versus host disease) and lack precise information on the myeloablative regimen used. Furthermore, our sample size is not very large and therefore the 95%CI of the reported proportions are wide. Wider implications of the findings Autologous or allogeneic HCT induces infertility in a very high percentage of patients. These findings support the need to prioritize sperm preservation before HCT also in cancer patients who are not candidate for HCT at first instance but who may need this treatment for failure of first line treatments. Trial registration number not applicable

Freezing chicken semen: Influence of base medium osmolality, cryoprotectants, cryoprotectant concentration, and cooling rate on post-thaw sperm survival

A classical chicken semen diluent (Lake's 7.1 diluent) was modified to have lowered osmolalities (ranging from 290 to 410 mOsm/kg). The modified medium with physiological osmolality of 325 mOsm/kg allowed cold storage of fresh semen for several days with very little loss of membrane integrity and motility, while high osmolalities inhibited motility. This modified medium was then used as base for freezing medium to test effects of the type and concentration of cryoprotective agent (CPA), and the cooling rate (CR). A number of CPAs (methylformamide, methylacetamide, dimethylformamide (DMF), dimethylacetamide (DMA), diethylformamide, and propylene glycol) were first compared by freezing semen with 0.6 mol/l of the respective CPA at a cooling rate of 250 °C/min. Post-thaw motility and membrane integrity were highest with DMA and DMF. Finally, in more detailed factorial experiments, semen from individual cocks or pooled semen was frozen using CRs of 4, 50, 250, and 440 °C/min and DMA concentrations ([DMA]) of 0.4, 0.6, 1.0, and 1.5 mol/l. Straws from each semen sample x treatment combination were divided for semen assessment at three different research groups for sperm motility, membrane integrity, kinked tails, and DNA fragmentation, using microscopy, computer assisted motility analysis, and flow cytometry. There were clear effects of both CR and [DMA] and their interaction. CRs 50 and 250 °C/min gave best post-thaw sperm performance. Higher DMA concentrations gave better post-thaw membrane integrity, but concentrations above 1.0 mol/l can decrease sperm velocity or even inhibit sperm motility. Therefore [DMA] may best be 0.6–1.0 mol/l at a CR of 50–250 °C/min.

Reproductive Indices of Naturally Mated and Artificially Inseminated Quail Hens (Coturnix japonica): Is Artificial Insemination of Japanese Quail Hens Feasible in a Local Setting?

Naturally mated and artificially inseminated Japanese quail (Coturnix japonica) hens’ eggs reproductive indices were investigated to evaluate natural mating or artificial insemination techniques for breeding quails in local farms. A total of 141 (108 hens and 33 cocks) matured quails of about 11 weeks of age were used for the experiment, of which 54 hens were inseminated with Citric-quail egg albumen extended semen collected from 15 fertile cocks. Another 54 hens were naturally mated by another 18 fertile cocks in a ratio of one cock to three hens (1:3). Eggs collected from both naturally mated and artificially inseminated birds were incubated for 18 days. The percentage of incubated eggs that were fertile when candled on the 7th and 12th day and hatch-out analysis after the 18th day of incubation tested fertility. Hatchability was tested by the percentage number of fertile eggs that hatched. Naturally mated and artificially inseminated hens’ eggs had a fertility of 84.0 % and 73.3 %, and a hatchability of 82.5 % and 85.5 %, respectively. The study revealed no significant difference in the frequency of fertile or hatched Japanese quail eggs (χ2 cal < χ critical at 5% or 1% level of probability). Naturally mated and artificially inseminated birds had relative fertile egg rates of 53.4 % and 46.6 %, respectively, and relative egg hatching rates of 52.5% and 47.5 %, respectively. Artificial insemination had the advantage of introducing the same volume of semen to more quail hens than a single cock in a natural mating process. However, natural mating was recommended over artificial insemination in the study area because of the technical constraints that required more workforce and amenities in the insemination process. The authors recommended research on developing restraint instruments and non-electrical semen storage methods for quail birds. The authors also proposed a restraint model for quail birds

The Effect of the Addition of Co-Q10 on the Preservation of Boar Semen at 17°C

Background: With the increasing application of artificial insemination technology in boars, breeding efficiency is largely determined by the quality of semen preservation. With the prolonged storage of semen at room temperature in vitro, excessive reactive oxygen species (ROS) damage organelles and plasma membranes and ultimately lead to a decline in semen quality. Methods: In order to improve the preservation of semen at 17°C, this experiment investigated the effect of different concentrations (0, 10, 15, 25, 35 µg/mL) of coenzyme Q10 (Co-Q10) in Modena dilution on the quality of boar semen during storage at 17°C. The sperm motility, mitochondrial activity, total antioxidant (T-AOC), superoxide dismutase (SOD) and malondialdehyde (MDA) were assessed at 17°C at different times (0, 1, 2, 3, 4, 5 d). Result: The results showed that the addition of Co-Q10 has a positive effect during the preservation of boar semen at 17°C. On the 5th day, the sperm motility of the 25 µg/mL Co-Q10 group was 73.80% and the mitochondrial activity was 82.94%, which was significantly higher (P less than 0.05) than that of other groups. In terms of antioxidant levels, the MDA content of the 25 µg/ml and 35 µg/ml Co-Q10 groups on the 5th day was significantly lower (P less than 0.05) than that of the other groups. At the same time, the 25 µg/ml group could effectively enhance the activity of antioxidant enzymes and the SOD and T-AOC were 17.33 U/mL and 3.17 U/mL on the 5th day significantly higher than the other groups (P less than 0.05). The results showed that the most appropriate concentration of Co-Q10 when storing boar semen at 17°C was 25 µg/ml.

Effect of magnetic field on productive and hysiological performance in broiler breeder

The experiment was conducted to determine the effect of magnetic field on the productive and physiological performance of Ross 308. The experimental flock consist of 60 hens and 10 cocks of broiler breeders, which randomly divided into four groups and distributed on the treatments with three replicates for each treatment. The treatments included the control treatment (T1), exposure the stored semen to 803 gauss magnetic field for 24 hours (T2), exposure the fertile eggs to 250 gauss magnetic field for 72 hours before entered the incubator (T3) and exposure the individual cages to 250 gauss magnetic field for 8 weeks (T4), and the performance traits of hen day egg production, egg weight, recorded from magnetization cages group and compared with control group. The results showed a significant decrease (P≤0.05) in fertility and hatchability of total eggs in T2 compared with other groups, and there was no significant difference in the hatchability of fertile eggs and embryonic mortality among treatments, egg weight and the hen day egg production. (HD).

Shielding effect of melatonin improves seminal quality and oxidative stress indices during chilled storage of ram semen.

Supplementation of antioxidant to semen extender maintains seminal quality by reducing oxidative stress during preservation time period. The main aim of this study was to investigate the effect of different concentrations of melatonin supplementation on liquid storage of Magra ram semen. This study was performed on adult Magra ram (n = 8), and seminal ejaculates (48) were collected and evaluated for various macroscopic and microscopic seminal quality parameters for further processing. After preliminary evaluation, ejaculates of each collection session were mixed and divided into four equal aliquots. All the aliquots were diluted (1:10) with Tris-citric fructose egg yolk extender contained sans melatonin served as control, whereas the other three aliquots were supplemented with 0.5, 1 and 2mM MLT which were grouped as MLT0.5, MLT1 and MLT2, respectively. Thereafter, the samples were stored at 4 ºC for 72h, and various seminal parameters (individual sperm progressive motility, viability, abnormalities, plasma membrane functionality) along with oxidative stress parameters (total antioxidant capacity (TAC), malondialdehyde (MDA)) were evaluated at 0, 24, 48 and 72h of preservation. The results indicated that the mean percent values for progressive sperm motility, viability, plasma membrane functionality and TAC were significantly higher (p < 0.05) in treated groups with the highest values in MLT1 group. Significantly lower (p < 0.05) percentage of total sperm abnormalities and MDA level was observed in treatment groups compared to control group. The results report that supplementation of 1mM melatonin efficiently maintains the seminal quality and ameliorates oxidative stress during preservation at 4 ºC to 72h.

Motility and oxidative stress of common carp Cyprinus carpio sperm during short-term storage

Short-term storage of semen is a simple and inexpensive procedure to deal with logistics of large-scale hatchery operations but can lead to oxidative stress and a significant decrease in sperm motility and velocity. To better understand the mechanisms responsible for the association of poor sperm quality with oxidative stress, in the present study we investigated the effect of refrigerated storage for 0, 24, 48, 72, and 144 h on sperm motility and curvilinear velocity and oxidant/antioxidant balance of common carp Cyprinus carpio. Percentage of motile sperm was significantly (p < 0.05) reduced stored for 72 h storage compared to that of fresh sperm. Sperm stored 144 h showed < 40% motility with an average velocity of 91.12 ± 10.4 µm s−1. A time-dependent increase in the level of the oxidative stress indices lipid peroxidation and carbonyl derivatives of proteins was observed. Increase (p > 0.05) in total superoxide dismutase was detected after 48 h, and glutathione reductase and glutathione peroxidase activity demonstrated a significant increase after 72 h. These results provide an additional tool for the development and improvement of short-term sperm preservation procedures commonly applied in aquaculture.

Changes in aquaporins mRNA expression and liquid storage at 17°C: A potential biomarker of boar sperm quality?

Artificial insemination (AI) for pigs relies on liquid storage of extended semen at 17°C, which preserves sperm quality and ensures its fertilizing capacity. Routine quality controls include the evaluation of sperm motility, viability and capacitation status. The physiological functions of all these features depend on transmembrane aquaporins (AQPs), proteins playing key roles in osmoadaptation. In this study, we made a relative quantification, using RT-qPCR, of the mRNA of several sperm AQPs in AI-liquid semen doses before and after a 48-hr incubation period, aiming to determine possible quantitative compromising expression changes during the process that could serve as a diagnostic tool. Our results showed a decrease in classical sperm motility variables (total and progressive motility and velocity) and sperm viability after 48-hr storage, whereas capacitation status increased overtime. mRNA expression increased in the orthodox AQP4 and AQP6 after 48-hr incubation, relative to control (0hr) and 24-hr time-points. Moreover, mRNA expression of aquaglyceroporins AQP3, AQP7 and AQP10 was higher after 48-hr incubation, confirmed by AQP7-protein validation using Western blot. Our results indicate that expression levels of AQPs-mRNA can change in ejaculated pig spermatozoa under conditions of ex-vivo incubation that could modify sperm homeostasis, suggesting it could eventually become a relevant molecular biomarker to assess the efficiency of liquid storage of pig semen.

Semen Collection, Cryopreservation and Artificial Insemination in Dogs

Today, the methods of artificial insemination, that have recently become a biotechnological phenomenon, have being used in dogs especially for those partners that are largely unable to mate because of differences in their body sizes. Artificial insemination is likely only by diluting the semen collected in order to protect it from the environmental conditions, by using some short- and long-term preservation methods and by transferring it into the females with proper techniques. Therefore, in this brief review, some data about the approach to the male at the time of semen collection, as well as the collection itself (digital manipulation, conic plastic rubber along with hand massage, and electrical stimulations (the electro-ejeculator)), dilution (Tes, Bes, Hepes, Pipes, Tris, Tes/Tris and commercial extenders, at 1:1-1:5 dilution rate), short (at +4 oC) - and long-term (in ampoule, pellet and straw) frozen storage (within liquid nitrogen, at -196 oC) of semen and artificial insemination methods (intravaginal catheter, Norwegian intrauterine catheter, endoscopic intrauterine and surgical intrauterine technique)) are given in dogs.

Potential Use of Tannin Extracts as Additives in Semen Destined for Cryopreservation: A Review.

Simple SummaryFreezing of semen used for artificial reproductive technologies (ART) affects the survival and vigour of sperm cells due to excessive production of reactive oxygen species (ROS) during the freezing and thawing processes. ROS plays a physiological role in sperm function but excessive ROS production from damaged sperm cells can hinder sperm’s motility and their ability to fertilise an oocyte. Tannins, a class of water-soluble plant polyphenols, are known to have antioxidant and other health-promoting effects and may serve as binders/acceptors to reduce the deleterious effects of excessive ROS produced during the freezing and thawing process. This review is the first to analyse the available data supporting the use of tannins as additives to semen extenders to improve the survival of cryopreserved spermatozoa during storage and after thawing. It is concluded that tannins and their derivatives have naturally protective properties with the potential to ameliorate sperm cell survival after freezing.Cryopreservation and storage of semen for artificial insemination (AI) result in excessive accumulation of reactive oxygen species (ROS). This leads to a shortened life span and reduced motility of spermatozoa post-thawing, with consequent impairment of their function. However, certain levels of ROS are essential to facilitate the capacitation of spermatozoa required for successful fertilisation. Tannins, as well-known antioxidant compounds, may act as ROS binders/acceptors/scavengers to inhibit the damaging effects of ROS. This review comprises an analysis of the semen cryopreservation protocol and health functions of tannins, as well as the effects of ROS on fresh and cryopreserved semen’s longevity and fertilisation. Additionally, we surveyed available evidence of the effects of tannin extract feed supplementation on male fertility. We furthermore interrogated existing theories on tannin use as a potential additive to semen extenders, its relationship with semen quality, and to what degree existing theories have been investigated to develop testable new hypotheses. Emphasis was placed on the effects of tannins on ROS, their involvement in regulating sperm structure and function during cryopreservation, and on post-thaw sperm motility, capacitation, and fertilising ability. The diverse effects of tannins on the reproductive system as a result of their potential metal ion chelation, protein precipitation, and biological antioxidant abilities have been identified. The current data are the first to support the further investigation of the incorporation of tannin-rich plant extracts into semen extenders to enhance the post-thaw survival, motility, and fertilising ability of cryopreserved spermatozoa.

Bal Arısı Spermasının Kısa ve Uzun Süreli Saklanmasıyla İlgili Güncel Yaklaşımlar

There are many studies on the storage of honey bee semen. Due to its unique structure, bee semen varies according to the storage period but is suitable for short-term storage conditions. However, in terms of long-term storage conditions, developments in mammals have not been caught. It is known that more studies are needed on this subject. The great advantages of semen storage are its effective role in eliminating the problems of inbreeding after a while in pure breed enterprises, increasing the yield characteristics and preventing gene pollution. To solve these problems, beekeepers have to buy breeding queens from abroad with very high costs, and the expected results cannot always be obtained from the bees brought. Especially considering the genetic risks faced by regional breeds in Turkey, it is necessary to preserve semen, to protect colonies in pure breed enterprises, as well as to develop genetically yield characteristics and to prevent inbreeding. It can also prevent the spread of diseases compared to natural mating by contributing to artificial insemination. By improving the storage conditions of honey bee semen, long-term use of semen from well-known colonies will be possible. Producers will be able to benefit from the advantages of artificial insemination at a cheaper cost, with the semen being sent to distant regions as in mammals under appropriate storage conditions. The purpose of this review is to give information about the storage conditions of honeybee semen.

Science-based quality control in boar semen production.

The ever-increasing understanding of sperm physiology, combined with innovative technical advances, continuously furthers the development of boar semen production management. These improvements pave the way for the future implementation of modified quality assurance concepts. This review provides an overview of current trends and new approaches in boar semen production, focusing on: the improvement of hygienic standards, alternatives to the use of antibiotics including the application of cold temperature storage and the utilization of antimicrobial additives, as well as the implementation of new quality control tools. Furthermore, the influence of dilution and temperature management, as well as new possibilities for an improvement of boar semen shipping and storage conditions are reviewed.

Assessment of Sperm Viability and Computer-Assisted Motility Analysis in Budgerigars (Melopsittacus undulatus): Effect of Several In Vitro Processing Conditions.

In order to preserve endangered psittacine species, more basic and applied research in reproductive biology is required. Assisted reproductive technologies such as artificial insemination play an important role in parrots species conservation programs to overcome the problem of infertile eggs and male infertility. The aim of this study was to define an effective in vitro protocol in order to standardize the sperm quality evaluation in psittacines, studying Melopsittacus undulatus as model species. Semen was collected from twenty adult males by massage technique from May to June. Sperm concentration was measured by the spectrophotometric method. Sperm quality (sperm membrane integrity (SMI), motility, and kinetic parameters) was assessed on fresh semen. Three different experimental protocols were performed to compare the effects of various processing conditions on SMI, motility, and kinetic parameters. In protocol 1, test was performed by Lake extender with three different pH, 7.4 versus 8.2 versus 8.4, and two different equilibration temperatures after dilution of fresh semen (4°C versus 25°C). In protocol 2, two dilution rates of semen after collection were valuated, 1 : 3 versus 1 : 4, as well as three different semen storage temperatures (4°C versus 25°C versus 38°C) before sperm motility analysis with the computer-assisted sperm analysis (CASA). In protocol 3, two different Makler chamber temperatures (38 versus 41°C) during motility analysis were tested. A significant progressive improvement in spermatozoa motility and kinetic parameters was registered with pH 8.4. Progressive motility and all kinetic parameters were higher at 4°C equilibration temperature. Straightness (STR) kinetic parameter was better with 1 : 4 dilution rate. Total motile sperm was higher in 41°C Makler chamber. In this study, for the first time, the effects of different processing protocols on psittacines seminal quality analysis were investigated. Significant differences conditioning the effectiveness of analysis protocols have been described.

The Protective Role of Melatonin in Sperm Cryopreservation of Farm Animals and Human: Lessons for Male Fish Cryopreservation.

Simple SummaryIn recent years, cryopreservation of fish sperm has been a rapidly evolving technique that contributes both to the improvement of genetic reproduction programs and the proper management of broodstock as well as to ensuring the viability of endangered species. However, this technique can cause significant damage to sperm, making the use of cryoprotectants and antioxidants in cryopreservation solutions imperative. The hormone melatonin has demonstrated positive effects on the cryopreservation of sperm in both farm animals and humans. Therefore, the plethora of research that has been conducted on animals and humans could be expanded to fish cryopreservation, making melatonin potentially a very promising alternative cryoprotectant.Cryopreservation is a technique that offers various advantages, especially in fish, among others, that makes the reproduction of species easier through a constant supply of sperm, synchronization of the gamete availability of both sexes, storage of semen for genetic improvement programs, reduction in the cost by eliminating the need to maintain male broodstock, and conserving the gametes of endangered species. However, freezing and warming procedures for cryopreservation lead to a reduction in the quality and viability of cryopreserved sperm because of oxidative stress. For this reason, the enrichment of extender media with antioxidants is a common method of cryopreservation of the semen of several fish species. Recently, many studies have been published for the protective role of antioxidants and especially of melatonin on male fertility preservation both in farm animals and humans, demonstrating the beneficial effects of melatonin as a sperm cryoprotectant. On the other hand, very few studies were conducted using melatonin as an antioxidant in different male fish species for semen cryopreservation. We conclude that the use of moderate concentrations of melatonin are beneficial to semen preservation, and the mechanisms through which melatonin acts positively on spermatozoa need to be further investigated to establish improvement protocols for cryopreservation in fish species.

Fertility comparison of frozen bull semen stored in cryogenic deep freezer (−152 °C) and LN2 container

The present study aimed to use cryogenic deep freezers that could be a feasible alternative for cryopreserved semen storage. A total of 284 straws from three Simmental bulls and 272 Simmental cows were used. The experimental group consisted of 151 semen straws that were stored at −152 °C for a week. Moreover, the control group consisted of 133 semen straws that were stored at −196 °C. Firstly, two samples per bull (n = 6) were examined in terms of sperm kinetic parameters by CASA. Furthermore, plasma membrane, acrosome integrity (PMAI) and high mitochondrial membrane potential (HMMP) were analyzed by flow cytometry. Then, artificial inseminations were performed on Simmental cows with 272 straws belonging to two groups. Then, 56th-day Non-return Rate (NRR56) was determined. All spermatological data were subjected to a linear mixed model. Chi-Square test was performed to NRR56 between storage temperature groups. Also, logistic regression analysis was used to examine the effect of bull, storage temperature and age of cows on pregnancy status. While age of cows was included in the final logistic regression model, effect of bull x storage temperature was not included because it was found as non-significant. The post-thaw PMOT and STR of cryopreserved bull semen, which was stored at −152 °C, had lower and statistically significant values (p < 0.05). However, frozen bull semen, which were stored at −152 °C, kept its fertility ability as which stored at −196 °C. Besides, NRR56 of semen stored at −152 °C and −196 °C were detected as 57.24% (83/145) and 55.91% (71/127), respectively (p > 0.05). Nevertheless, these results should be supplemented with more pre-freezing and post-thaw sperm quality analyses and more fertility data for increasing the accuracy of the method.

Effects of antioxidant use on semen storage in honey bees

Although there are many studies for the storage of semen in honey bees, the desired success has not been achieved, and more study is needed in this area. It has been reported that the percentage of bee egg-laying in queen bees fertilized with stored semen, especially in long-term storage conditions is below the expected rate despite the viability rate. The reason for this situation is that honey bee spermatozoa are negatively affected by freezing processes due to their very fragile and sensitive structure. However, due to natural mating, honey bee semen can remain healthy for years in the queen bee's spermatheca. It is known that there are many enzymatic antioxidants and special proteins in the spermatheca, as well as a suitable environment for spermatozoa. Manipulating made during the stored of honey bee semen has adverse effects on spermatozoon function and fertility. In particular, current antioxidant substances against cold shock, which are among the damages that occur during freezing of semen, are being investigated, and their protective effects on semen are determined. For this purpose, it is aimed to improve the storage conditions of honey bee semen by using substances with antioxidant properties. The purpose of this review is to give information about the use of antioxidant substances in the storage of honey bee semen.

The Effect of the Liquid Nitrogen Level on the Temperature in a Semen Storage Tank

The temperature in a liquid nitrogen storage tank is critical to maintain the viability of bovine semen stored within the tank. The objective was to determine the relationship of the temperature gradient in the neck of a tank to the level of liquid nitrogen in the tank. Using an electronic thermometer, temperature was measured at three locations: a) 2 cm below the top of the neck, b) 7 cm below the top of the neck (and below the frost line), and c) 2 cm above the level of liquid nitrogen. Liquid nitrogen volume in the tank was incrementally decreased from 18 L to 6 L.

A pilot study about assisted reproduction in harpy eagles (Harpia harpyja) in the course of species conservation including collection, storage, and analysis of semen

Currently, 52% of all raptor species demonstrate a decreasing population tendency, and the American harpy eagle (Harpia harpyja) has been categorized as “near threatened” by the IUCN. Habitat loss, persecution, and subsequent reduction of genetic diversity are regarded as major threats to the world's strongest eagle. Captive breeding and reintroduction into protected habitats are approaches of species conservation projects, but captive propagation is difficult due to low ex-situ numbers and scarce successful breeding pairs.The aim of the present study was to collect, analyze, and store semen from harpy eagles and to use aliquots for artificial insemination to increase the number of offspring and to include more individuals into the ex-situ gene pool. First, semen collection and semen availability were assessed in four males during the course of 1 year in European zoos. Second, these experiences were transferred to ex-situ breeding programs in Brazilian zoos to attempt semen collection in 13 male eagles.Semen collection was successful in 51.7% of the attempts and in 8/13 males (individual success rates 20–100%) using electro-stimulation. Most commonly, whey-like to milky, whitish semen samples were collected, regularly containing urate impurities (67.7%). The median semen volume was 106 μl and the median sperm concentration 5,000 sperm/μl (750–22,500 sperm/μl). Mean values for pH were 6.7, for sperm motility 27.7 ± 22.6%, for progressive motility 2.9 ± 5.6%, and for sperm viability 46.6 ± 16.3%. Using semen extenders, a sperm motility of 8% was maintained for 27 h in the refrigerator. Artificial insemination was performed in one female, but the success of fertilization could not be assessed due to egg destruction.In this study, methods for assisted reproduction were refined for use in harpy eagles, and the first semen samples were evaluated as a start to establish species-specific orientation values.

Effects of low-density lipoprotein extracted from egg yolk on preservation of chilled canine semen (4oC)

Low-density lipoprotein (LDL) derived from hen egg yolk has been considered an important component in the storage of canine semen; however, in Vietnam, there have not been many studies regarding this topic. Therefore, this study aimed to evaluate the effectiveness of LDL in the preservation of canine chilled semen in Vietnam. The assessment of semen samples was based on four criteria: storage time, motility, absolute viability, and integrity of acrosomal membrane at motility rate 0.9; 0.5 and 0.3. The semen samples collected were divided into three equal parts, each was then diluted with three different extenders and stored at 4oC. The extenders were LDL + basic extender (LDL), egg yolk + basic extender (EY), and basic extender (C). The storage time of canine semen was recorded from the beginning of monitoring until the motility decreased to 0.3. The storage time of the three extenders was in the order of EY &gt; LDL &gt; C, with 108 h, 60 h, and 48 h, respectively. The absolute viability of fertilizable sperm (Sa5) in the extender was EY (768), LDL (423) and C (280), which was significant (P &lt; 0.001). The percentage of viable sperm with intact acrosome membrane at motility time A = 0.9 in LDL was 59.31%, higher than that in EY (30.99%). In the storage period from A = 0.9 to A = 0.5, the acrosome loss percentage of both EY and LDL decreased equally (7.30% and 7.23%), but the storage time of EY (84 h) was longer than that of LDL (48 h). In conclusion, the EY gave a longer storage time, higher absolute viability and longer maintain percentage of viable sperms and intact acrosome membrane compared to the LDL. However, within the first hours of storage, the percentage of viable sperm and intact acrosome membrane in the EY was lower than that in the LDL. Therefore, to preserve canine semen for a short time (less than 48 h), the LDL extender should be used for better effectiveness.

Effects of boar sperm antioxidant supplementation on fertility

Use of cooled stored boar semen for artificial insemination accounts for 99% of inseminations in pork production sectors worldwide. The aim of the present study was to examine use of GameteGuard®-CP, a natural plant derived blend of antioxidants, supplemented in one commercially available semen extender on sperm quality, conception rate, farrowing rate, and litter size. Ejaculates from 16 commercial Duroc boars were used in a split-ejaculate single-sire artificial insemination study. Sperm were evaluated for motility, viability, acrosome integrity, membrane stability, mitochondrial membrane potential, DNA intactness, and total antioxidant reactivity on both day 1 and 4 subsequent to ejaculate collection. Sows were inseminated after a treatment regimen was imposed to synchronize time of ovulation (n = 1476) among females using sperm collected 4 days subsequent to ejaculate collection extended in AndroStar Plus® or GameteGuard®-CP supplemented extender. At day 4 post-ejaculate collection, there was maintenance of sperm viability, membrane stability, acrosome intactness, mitochondrial membrane potential, and DNA intactness (P < 0.01) when there was use of GameteGuard®-CP treatments. When control samples were used, there were lesser (P < 0.01) values from day 1–4 for all variables evaluated. Conception rate and farrowing rates were 7.4% and 9.7% greater, respectively (P < 0.05), but there was no difference as a result of GameteGuard®-CP-treatment in mean litter outcomes. The results in the present study indicate antioxidant supplementation using GameteGuard®-CP maintains sperm quality during cool sperm storage resulting in improved conception and farrowing rates when using semen stored in the cooled state for as long as 4 days.