A functional assessment of the sperm membrane: a multi-species approach

A functional sperm membrane is essential for many of the processes which lead to the fertilisation of an oocyte, but this membrane is susceptible to oxidative damage with increasing duration of storage in liquid semen or during the cryopreservation process. The objectives of this thesis were to examine the effects of storage temperature, catalase supplementation and sperm number on the membrane function of liquid stored bull semen, sperm membrane protein profile on the membrane integrity of liquid stored boar semen and stabilisation of the stallion sperm membrane using cholesterol, prior to cryopreservation, on the membrane function of frozen-thawed stallion semen. Sperm progressive motility was assessed by microscopy, glucose consumption and total antioxidant capacity were assessed using commercial kits, and viability, oxidative stress, mitochondrial activity, acrosomal status and membrane fluidity were assessed by flow cytometry. Conception rates in dairy cows were assessed as the 60 day non-return rate in a field study assessing the effects of lowering the sperm number in liquid bull semen, which included a frozen-thawed semen treatment. In liquid bull semen, storage at temperatures above 22 °C had reduced motility and membrane integrity (P < 0.05), in comparison with storage at 5 to 22 °C. Catalase supplementation had no effect on oxidative stress in liquid semen (P > 0.05). Reducing the sperm number in liquid semen insemination doses reduced ROS generation and glucose consumption, and increased viability (P < 0.05), however, storage at 4 and 3 × 106 sperm per dose had a reduced non-return rate on Day 2 of storage in comparison to the frozen-thawed semen treatment (P < 0.01). In liquid boar semen, the sperm membrane and seminal protein profiles differed between boars of high and low sperm membrane integrity (P < 0.05). Peroxiredoxin 5 and α-mannosidase were found to have a greater expression in sperm from boars of high membrane integrity (P < 0.05), while fibronectin, thrombospondin, AQN-1 and PSP-1 had a greater expression in sperm from boars of low membrane integrity (P < 0.05). In frozen-thawed stallion semen, the addition of cholesterol, prior to cryopreservation, increased sperm viability and membrane integrity, and reduced the generation of the superoxide anion, post-thaw (P < 0.05). In conclusion, this thesis provides new insights into sperm cell function in the bull, boar and stallion. Storage of liquid bull semen at lower sperm numbers is beneficial in reducing oxidative stress, but lowers non-return rates on Day 2 of storage. Boars differing in field fertility can be differentiated based on their sperm membrane protein profile while the addition of cholesterol to stallion semen, prior to cryopreservation, is beneficial to membrane function and semen quality, post-thaw

A functional assessment of the sperm membrane: a multi-species approach

A functional sperm membrane is essential for many of the processes which lead to the fertilisation of an oocyte, but this membrane is susceptible to oxidative damage with increasing duration of storage in liquid semen or during the cryopreservation process. The objectives of this thesis were to examine the effects of storage temperature, catalase supplementation and sperm number on the membrane function of liquid stored bull semen, sperm membrane protein profile on the membrane integrity of liquid stored boar semen and stabilisation of the stallion sperm membrane using cholesterol, prior to cryopreservation, on the membrane function of frozen-thawed stallion semen. Sperm progressive motility was assessed by microscopy, glucose consumption and total antioxidant capacity were assessed using commercial kits, and viability, oxidative stress, mitochondrial activity, acrosomal status and membrane fluidity were assessed by flow cytometry. Conception rates in dairy cows were assessed as the 60 day non-return rate in a field study assessing the effects of lowering the sperm number in liquid bull semen, which included a frozen-thawed semen treatment. In liquid bull semen, storage at temperatures above 22 °C had reduced motility and membrane integrity (P < 0.05), in comparison with storage at 5 to 22 °C. Catalase supplementation had no effect on oxidative stress in liquid semen (P > 0.05). Reducing the sperm number in liquid semen insemination doses reduced ROS generation and glucose consumption, and increased viability (P < 0.05), however, storage at 4 and 3 × 106 sperm per dose had a reduced non-return rate on Day 2 of storage in comparison to the frozen-thawed semen treatment (P < 0.01). In liquid boar semen, the sperm membrane and seminal protein profiles differed between boars of high and low sperm membrane integrity (P < 0.05). Peroxiredoxin 5 and α-mannosidase were found to have a greater expression in sperm from boars of high membrane integrity (P < 0.05), while fibronectin, thrombospondin, AQN-1 and PSP-1 had a greater expression in sperm from boars of low membrane integrity (P < 0.05). In frozen-thawed stallion semen, the addition of cholesterol, prior to cryopreservation, increased sperm viability and membrane integrity, and reduced the generation of the superoxide anion, post-thaw (P < 0.05). In conclusion, this thesis provides new insights into sperm cell function in the bull, boar and stallion. Storage of liquid bull semen at lower sperm numbers is beneficial in reducing oxidative stress, but lowers non-return rates on Day 2 of storage. Boars differing in field fertility can be differentiated based on their sperm membrane protein profile while the addition of cholesterol to stallion semen, prior to cryopreservation, is beneficial to membrane function and semen quality, post-thaw

A comparison of semen diluents on the in vitro and in vivo fertility of liquid bull semen

The aim of this study was to assess the effect of semen diluent on calving rate (CR) following artificial insemination with liquid bull semen stored for up to 3 d postcollection. In experiment 1, the effect of storing liquid semen maintained at a constant ambient temperature in 1 of 7 different diluents [Caprogen (homemade), OptiXcell, BioXcell, BullXcell, INRA96, NutriXcell, or AndroMed (all commercially available)] on total and progressive motility was assessed on d 0, 1, 2, and 3 postcollection. In experiment 2, the field fertility of liquid semen diluted in Caprogen, BioXcell, or INRA96 and inseminated on d 1, 2, or 3 postcollection was assessed in comparison to frozen-thawed semen (total of n = 19,126 inseminations). In experiment 3, the effect of storage temperature fluctuations (4 and 18 C) on total and progressive motility following dilution in Caprogen, BioXcell, and INRA96 was assessed on d 0, 1, 2, and 3 postcollection. In experiment 1, semen stored in Caprogen, BioXcell, and INRA96 resulted in the highest total and progressive motility on d 1, 2, and 3 of storage compared with OptiXcell, BullXcell, NutriXcell, and AndroMed. In experiment 2, an effect of diluent on CR was found as semen diluted in BioXcell had a lower CR on d 1, 2, and 3 of storage (46.3, 35.4, and 34.0%, respectively) in comparison with Caprogen (55.8, 52.0, and 51.9%, respectively), INRA96 (55.0, 55.1, and 52.2%, respectively), and frozen-thawed semen (59.7%). Effects were found of parity, cow fertility sub-index, as well as the number of days in milk on CR. In experiment 3, when the storage temperature of diluted semen fluctuated between 4 and 18 C, to mimic what occurs in the field (nighttime vs. daytime), BioXcell had the lowest total and progressive motility in comparison to Caprogen and INRA96. In conclusion, diluent significantly affected sperm motility when stored for up to 3 d. Semen diluted in INRA96 resulted in a similar CR to semen diluted in Caprogen and to frozen-thawed semen, whereas that diluted in BioXcell resulted in a decreased CR. Consistent with this finding, semen diluted in BioXcell was less tolerant of temperature fluctuations than that stored in Caprogen or INRA96. Given that it can be used directly off the shelf, INRA96 may be a suitable alternative to Caprogen for the storage of liquid bull semen