Cryopreservation compromises the capacity of sperm fertilizing due to a series of alterations in the structure and physiology of the sperm. The use of antioxidants, such as melatonin, added to freezing media, may help to reduce sperm cryoinjury. To test the effect of melatonin on Bulldog (Canis lupus familiaris) sperm cryosurvival, spermatozoa were diluted in a standard freezing medium and cooled to 5°C. Then, more freezing medium was added to obtain 200 × 10⁶ cells/mL, and 5% glycerol. Diluted spermatozoa were treated with melatonin (0.0, 0.0005, 0.002, and 0.0035 mol/L), and packaged in 0.25 mL straws, which were further cooled to −5°C before freezing in liquid nitrogen. Thawing was carried out at 70°C for 5 s, and the progressive motility, viability, plasma membrane integrity, acrosome integrity, capacitation status, and plasma membrane fluidity of the spermatozoa (at 37°C) were assessed. Data were analyzed using ANOVA to detect differences between the melatonin doses. There were statistical differences (P < 0.05) in the percentage of sperm having hyper-fluid membranes, intact acrosome, capacitated acrosome-intact, and acrosome-reacted. The values for the high melatonin doses (0.002 and 0.0035 mol/L) were better than for the low melatonin doses (0.0 and 0.0005 mol/L). In conclusion, 0.002 and 0.0035 mol/L of melatonin improved the cryosurvival of sperm from male bulldogs. Lay summary: Preservation of sperm by freezing enables breeding of individuals geographically separated; protocols for the dog may be used to preserve the semen from threatened wild canids. To improve fertility of female dogs that become pregnant with frozen and then defrosted sperm, these cells must survive that process which can be damaging whilst keeping their ability to fertilize. Antioxidants are substances capable of retarding or preventing the oxidation of any oxidizing substrate such as lipids, proteins, and DNA, which are structural compounds of the sperm. The use of antioxidants, added to freezing media, may provide the sperm the capacity to neutralize oxidative compounds, such as reactive oxygen species, produced during the freezing and thawing process. In this work we tested different levels of melatonin, a natural antioxidant, on dog (English Bulldog) sperm survival and quality after freezing. We found that adding melatonin to the freezing media improved sperm quality after thawing.

In mammals, the pineal hormone melatonin is the most powerful pacemaker of the master circadian clock and is responsible for reproduction in seasonal breeders. It is also well known that melatonin and its metabolites play antioxidant roles in many tissues, including reproductive cells. Melatonin synthesis and secretion from the pineal gland occurs during scotophase (the dark phase during a day–night cycle), while its inhibition is observed during photophase (period of light during a day–night cycle). Short-day breeders, such as goats, are stimulated to breed in a manner dependent on high endogenous levels of melatonin. This hormone can be synthesized in various extra-pineal tissues, such as retina, gastrointestinal tract, ovaries, and testis, with its main function being as a local antioxidant, given that melatonin and its metabolites are potent scavengers of reactive oxygen and nitrogen species. Moreover, it has been reported that some functions of melatonin can be exerted through plasma membrane and intracellular receptors expressed in the male reproductive system, including germ cells, immature and mature spermatozoa. It has been shown that melatonin may enhance gamete cryosurvival mainly by its addition into the media and/or in exogenous melatonin treatments in several species. In the present review, the physiological effects of endogenous melatonin in mammals are described, with a deeper focus on caprine reproduction. Additionally, results from recent investigations on the roles of exogenous melatonin aimed at improving the reproductive efficiency of goat bucks are discussed. There are contradictory findings and a limited amount of research available in the field of goat sperm cryopreservation associated with the use of melatonin. Understanding and improving goat reproduction and production is essential for many marginalized human populations around the world who directly depend on goats to maintain and improve their lifestyle.

Actual loss of lizard biodiversity continues, even with the implementation of conventional conservation programs. An approach including assisted reproductive techniques such as sperm cryopreservation may contribute to the management of endangered species. We developed a method for sperm cryopreservation in sceloporine lizards and compared the response among the studied species. Prior to the mating season, we obtained semen from adult males of Sceloporus aeneus (n = 21), Sceloporus grammicus (n = 20) and Sceloporus torquatus (n = 21) via pressure of the genital papilla. Volume and sperm concentration were measured before semen dilution in a Tris–egg yolk (TEY) medium to evaluate progressive motility, sperm viability, morphology, plasma membrane and acrosome integrity. Then, we cooled the remaining volumes to 5°C at a rate of 0.1°C per minute to incorporate glycerol (8% v/v) in two fractions. Immediately afterwards, we placed 40 μl of the mix on solid CO₂ to form pellets and immersed them in liquid nitrogen for storage. We thawed the pellets at 29°C for 3 minutes and diluted them 1:1 (v/v) in TEY medium to assess sperm quality. We found a positive relationship between body weight and seminal volume in S. grammicus and S. torquatus and a negative correlation with sperm concentration in S. grammicus (P < 0.05). Moreover, we observed that the freezing–thawing process decreased sperm quality in the three species, mostly affecting motility and viability. However, S. torquatus and S. aeneus showed a higher sperm tolerance than S. grammicus.