Protein Identification of Spermatozoa and Seminal Plasma in Bottlenose Dolphin (Tursiops truncatus).

Proteins play an important role in many reproductive functions such as sperm maturation, sperm transit in the female genital tract or sperm-oocyte interaction. However, in general, little information concerning reproductive features is available in the case of aquatic animals. The present study aims to characterize the proteome of both spermatozoa and seminal plasma of bottlenose dolphins (Tursiops truncatus) as a model organism for cetaceans. Ejaculate samples were obtained from two trained dolphins housed in an aquarium. Spermatozoa and seminal plasma were analyzed by means of proteomic analyses using an LC-MS/MS, and a list with the gene symbols corresponding to each protein was submitted to the DAVID database. Of the 419 proteins identified in spermatozoa and 303 in seminal plasma, 111 proteins were shared by both. Furthermore, 70 proteins were identified as involved in reproductive processes, 39 in spermatozoa, and 31 in seminal plasma. The five most abundant proteins were also identified in these samples: AKAP3, ODF2, TUBB, GSTM3, ROPN1 for spermatozoa and CST11, LTF, ALB, HSP90B1, PIGR for seminal plasma. In conclusion, this study provides the first characterization of the proteome in cetacean sperm and seminal plasma, opening the way to future research into new biomarkers, the analysis of conservation capacity or possible additional applications in the field of assisted reproductive technologies.

Characterization of Bottlenose Dolphin (Tursiops truncatus) Sperm Based on Morphometric Traits.

Bottlenose dolphin (Tursiops truncatus) males follow many reproductive strategies to ensure their paternity. However, little is known about the sperm traits, including morphometric features, that contribute to their reproductive success. Our aim was to study dolphin sperm morphometry (a total of 13 parameters) in two adult males to evaluate (i) presumptive sperm subpopulations, (ii) the correlation of sperm morphometry with testosterone levels and (iii) the effect of refrigerated storage on the sperm morphometry. Sperm populations were classified into four principal components (PCs) based on morphometry (>94% of cumulative variance). The PCs clustered into two different sperm subpopulations, which differed between males. Furthermore, the levels of serum testosterone were positively correlated with the length of the midpiece but negatively correlated with head width and the principal piece, flagellum and total sperm lengths. Most of the sperm morphometric parameters changed during the storage period (day 1 vs. day 7), but only the principal piece length was affected by the storage temperature (5 °C vs. 15 °C). This is the first study to identify dolphin sperm subpopulations based on morphometry and the influence of serum testosterone and refrigeration on sperm morphometry.

Sperm collection by electroejaculation in small ruminants: A review on welfare problems and alternative techniques.

There are different perspectives on whether there should be use of electroejaculation (EE) for semen collection because it can be stressful and painful for the males when this technique is imposed. In the present review it is examined 1) the effects of EE on animal welfare and semen quality in domestic and wild small ruminants, 2) benefits and limitations of administering anaesthetics and sedatives prior to EE, 3) advantages/disadvantages of transrectal ultrasonic-guided massage of the accessory sex glands (TUMASG) as an alternative to EE, and 4) benefits of administering hormones, such as oxytocin or PGF2α analogues (which stimulate the contractility of the male accessory sex glands), prior to EE and TUMASG. In general, the administration of anaesthetics, sedatives or hormones reduces the pain and stress caused by EE, and can improve sperm quality, but results may vary depending on the species. The use of anaesthetics is, however, not devoid of risks and pre-EE administration of sedatives, or oxytocin or PGF2α analogues, can aid sperm collection mitigate risks. The TUMASG is less stressful than EE, but its effectiveness varies greatly among species, and it can only be performed by trained personnel. Prior administration of the hormones may also result in a reduction in the period needed to induce ejaculation with use of TUMASG procedures.

Sperm collection by transrectal ultrasound-guided massage of the accessory sex glands is less stressful than electroejaculation without altering sperm characteristics in conscious goat bucks.

In anesthetized non-domestic ruminants transrectal ultrasound-guided massage of the accessory sex glands (TUMASG) is an alternative method to collect semen slightly less stressful than electroejaculation (EE). However, some sperm characteristics are better when semen is collected with EE than with TUMASG. As anesthesia reduces the response to stressors, the advantages of TUMASG may be reduced in anesthetized animals, and thus, TUMASG may be even more advantageous in conscious animals. Therefore, the aim of the present study was to compare the stress response and the characteristics of the sperm collected with TUMASG and EE in conscious goat bucks. Semen was collected in 10 bucks with both procedures. During each procedure, the time required for ejaculation, the number of electric pulses applied and the number of vocalizations were recorded. Rectal temperature, heart rate, serum cortisol concentration, biochemical and hematological parameters were measured before and after each procedure. Sperm characteristics [ejaculated volume, sperm concentration, sperm mass motility (scale 0-5), sperm vigor (scale 0-5), the percentages of motile and progressive motile sperm, of sperm vitality, of sperm with plasma membrane integrity, and with acrosome damage and morphological abnormalities] were also determined. Electroejaculation required more electric pulses than TUMASG (P < 0.0001), but TUMASG took more time than EE (P < 0.0001). The EE provoked more vocalizations (P = 0.02) and a greater increase of cortisol concentrations than TUMASG (P = 0.04). Heart rate also tended to be greater with EE than with TUMASG (P = 0.07). The sperm characteristics did not differ between TUMASG and EE. In conclusion, TUMASG was less stressful and probably less painful than EE without affecting the semen quality. Thus, although it required more time, TUMASG is an alternative procedure to decrease the welfare concerns raised by EE in conscious goat bucks.

Oxytocin administration before sperm collection by transrectal ultrasonic-guided massage of the accessory sex glands in mouflons and bucks.

Transrectal ultrasonic-guided massage of the accessory sex glands (TUMASG) is an alternative technique for sperm collection which decreases negative animal welfare concerns of electroejaculation. However, quality of collected sperm with TUMASG is less than that collected by electroejaculation. Utilisation of TUMASG may not, however, induce strong muscular contractions, so administration of oxytocin before the procedure may improve sperm quality. Therefore, two studies were designed to determine if administration of oxytocin before TUMASG improves the procedure and quality of the collected semen. The first study was performed with anaesthetised mouflons, and the second with non-anaesthetised Gabon bucks. Time length, number of electric pulses applied and numbers of vocalizations were recorded, and sperm characteristics were analysed. The number of electrical pulses and sperm characteristics did not differ with oxytocin administration in mouflons. However, oxytocin administration decreased time length (P=0.02) and tended to decrease the number of electric pulses needed for induction of ejaculation (P=0.08) in bucks. The number of vocalizations and sperm variables did not differ when the two procedures were used. In conclusion, oxytocin administration before TUMASG shortened the procedure and tended to decrease the number of electrical pulses needed to induce ejaculation in conscious bucks, thus, decreasing welfare concerns for the animals in which TUMASG is used. Therefore, its routine inclusion in this species should be considered.

Morphological study of boar sperm during their passage through the female genital tract.

Once deposited in the female tract, sperm face a series of challenges that must be overcome to ensure the presence of an adequate normal sperm population close to the site of fertilization. Our aim was to evaluate the influence of the uterine milieu on boar sperm morphology. In experiment 1, sperm morphology was evaluated in the backflow (60 min after insemination) and within the uterotubal junction (UTJ) (collected ~24 h after insemination) following intrauterine sperm deposition (n = 6) and compared with the morphology of the sperm in the insemination dose. In experiment 2, the influence of the uterine fluid (UF) on sperm morphological modifications was evaluated. For this purpose, ejaculated (n = 4) and epididymal (n = 4) sperm were in vitro incubated with or without UF for 2 and 24 h. In both experiments, sperm were classified as normal, having a cytoplasmic droplet (proximal or distal) or having tail defects. The results of experiment 1 pointed to an increase in morphologically abnormal sperm collected in the backflow (27.70%) and a reduction of the same in the UTJ (2.12%) compared with the insemination dose (17.75%) (P < 0.05). In experiment 2, incubation of ejaculated sperm with UF did not provoke any morphological modifications; however, when epididymal sperm were incubated with UF, a pronounced increase in the percentage of normal sperm was evident after 24 h compared with the initial dose (from 25.77% to 53.58%, P < 0.05), mainly due to distal cytoplasmatic droplet shedding (53.22 vs. 20.20%). In conclusion, almost all the sperm that colonize the UTJ had a normal morphology, with part of the abnormal sperm having been discarded in the backflow and part selected/modified on their way to the oviduct. UF seems to influence cytoplasmic distal droplet removal, as demonstrated previously in seminal plasma.