Sperm cryopreservation: current status and future developments.

The cryopreservation of spermatozoa is an important reproductive technology for the preservation of fertility in man and animals. Since the serendipitous discovery of glycerol as an effective cryoprotectant in 1947, sperm cryopreservation has undergone many changes in terms of the freezing methods employed, the rates at which samples are frozen and thawed, and the media used to preserve sperm functionality and DNA integrity. An extensive literature survey has been conducted addressing the cryoprotectants employed for both animal and human semen and the freezing protocols utilised. The results indicate that glycerol remains the dominant cryoprotective agent, usually incorporated into a balanced salt solution containing energy substrates, buffers, osmolytes and protein in the form of human serum albumin (human) or skimmed milk (animal). Realisation that some of the damage observed in cryostored cells involves the generation of reactive oxygen species during the thawing process, has prompted many studies to assess the relative merits of incorporating antioxidants into the cryopreservation media. However, in the absence of systematic comparisons, there is currently no consensus as to which antioxidant combination might be the most effective. Utilising our fundamental understanding of cryodamage to optimise cryopreservation protocols for each species will be important in the future.

Addition of Vitamin C Mitigates the Loss of Antioxidant Capacity, Vitality and DNA Integrity in Cryopreserved Human Semen Samples.

Cryopreservation of human spermatozoa is a necessity for males suffering from infertility who cannot produce fresh semen for insemination. However, current ART cryopreservation protocols are associated with losses of sperm motility, vitality and DNA integrity, which are thought to be linked to the induction of oxidative damage and the toxic properties of commercial cryoprotectants (CPAs). Preventing or mitigating these losses would be hugely beneficial to sperm survival during ART. Therefore, in this in vitro investigation, lipid peroxidation, production of reactive oxygen species, movement characteristics, antioxidant capacity, vitality, and DNA integrity were examined in semen samples both pre- and post-cryopreservation with CPA supplementation. The findings revealed a 50% reduction in antioxidant capacity with CPA addition, which was accompanied by significant increases in generation of reactive oxygen species and formation of lipid aldehydes. These changes were, in turn, correlated with reductions in sperm viability, motility and DNA integrity. Antioxidant supplementation generated bell-shaped dose-response curves with both resveratrol and vitamin C, emphasising the vulnerability of these cells to both oxidative and reductive stress. At the optimal dose, vitamin C was able to significantly enhance vitality and reduce DNA damage recorded in cryopreserved human spermatozoa. An improvement in sperm motility did not reach statistical significance, possibly because additional pathophysiological mechanisms limit the potential effectiveness of antioxidants in rescuing this aspect of sperm function. The vulnerability of human spermatozoa to reductive stress and the complex nature of sperm cryoinjury will present major challenges in creating the next generation of cryoprotective media.

Analysis of sperm separation protocols for isolating cryopreserved human spermatozoa.

Abstract: Sperm cryopreservation is a valuable tool for the long-term preservation of male fertility. Thus, determining the optimal technique for isolating spermatozoa post-thaw is vital to ensure recovery of the highest quality spermatozoa with minimal iatrogenic damage. This not only enhances the chances of successful conception but also reduces the risk of genetic damage in the embryo. To address this issue, human semen samples were cryopreserved using a slow freezing protocol and Quinn's Advantage™ Sperm Freeze medium. The samples were subsequently thawed and subjected to three types of sperm isolation procedures: direct swim-up, density gradient centrifugation, and electrophoretic separation using the Felix™ device. Cryopreservation led to the anticipated loss of sperm motility and vitality in association with increases in lipid peroxidation and DNA damage. Following sperm selection, all three isolation techniques resulted in an increase in sperm motility which was particularly evident with the swim-up and Felix™ procedures. The latter also significantly improved sperm vitality. There were no differences between sperm separation techniques with respect to morphology, and mitochondrial reactive oxygen species generation remained essentially unchanged when cell vitality was taken into account. By contrast, major differences were observed in DNA integrity and lipid aldehyde formation, where Felix™ isolated cells exhibiting significantly less DNA damage than the other isolation procedures as well as lower levels of 4-hydroxynonenal formation. Electrophoretic sperm isolation, therefore, offers significant advantages over alternative separation strategies, in terms of the quality of the gametes isolated and the time taken to achieve the isolation. Lay Summary: Long-term storage of sperm is vital to assisted reproductive technology because it permits the preservation of fertility that might be compromised as a result of factors such as chemotherapy or vasectomy. This goal can be achieved via cryopreservation - the freezing of cells to -196°C. When the sperm are subsequently required for conception, they must be carefully separated from the cryopreservation medium in a manner that maximizes the chances of successful conception and minimizes the risk of genetic defects in the offspring. In this paper, three isolation techniques were compared for their ability to separate ideal sperm from semen and media following cryopreservation. It was found that cryopreservation led to lower levels of motility and vitality and created higher levels of DNA and cell membrane damage. Of the three techniques compared, only cells separated on the basis of their size and electric charge (electrophoretic isolation) exhibited significantly lower levels of DNA fragmentation.

The intake pattern and feed preference of layer hens selected for high or low feed conversion ratio.

Feed accounts for the greatest proportion of egg production costs and there is substantial variation in feed to egg conversion ratio (FCR) efficiency between individual hens. Despite this understanding, there is a paucity of information regarding layer hen feeding behaviour, diet selection and its impact on feed efficiency. It was hypothesised that variation in feed to egg conversion efficiency between hens may be influenced by feeding behaviour. For this experiment, two 35-bird groups of ISA Brown layers were selected from 450 individually caged hens at 25-30 weeks of age for either low FCR < 1.8 ± 0.02 (high feed efficiency (HFE) or high FCR > 2.1 ± 0.02 (low feed efficiency (LFE)). For each of these 70 hens, intake of an ad-libitum mash diet at 2-minute time intervals, 24 h a day, for 7 days was determined alongside behavioural assessment and estimation of the selection of components of the mash. The group selected for HFE had a lower feed intake, similar egg mass and associated lower FCR when compared with the LFE group. Whilst feed intake patterns were similar between HFE and LFE hens, there was a distinct intake pattern for all layer hens with intake rate increasing from 0300 to 1700 h with a sharp decline to 2200 h. High feed efficiency hens selected a diet with 25% more ash and 4% less gross energy than LFE hens. The LFE hens also spent more time eating with more walking events, but less time spent resting, drinking, preening and cage pecking events as compared with HFE hens. In summary, there was no contrasting diurnal pattern of feed consumption behaviour between the groups ranked on feed efficiency, however high feed efficiency hens consumed less feed and selected a diet with greater ash content and lower gross energy as compared with LFE hens. Our work is now focused on individual hen diet selection from mash diets with an aim of formulating precision, targeted diets for greater feed efficiency.