No abstract

Semen preservation remains challenging in the camel industry. The objective of the present study was to compare 3 different extenders for chilled storage of Bactrian camel semen. Semen (n = 9 ejaculates) was collected from camel bulls (n = 2) manually using artificial vagina. Good neat semen, as far as mass vibration concerns, was equally distributed into 3 double-wall vessels filled with 35°C water. The 3 extenders used in the present study were Hashi, Green buffer (IMV, L’Aigle, France), and INRA 96 (IMV). The Hashi extender consisted of Tris, 2.6%; citric acid, 1.35%; glucose, 0.9%; fructose, 0.9%; penicillin G sodium, 1000 IU mL−1; streptomycin sulfate, 1000 mg mL−1 supplemented with 20% plasma egg yolk and 20% camel skim milk; osmolality of 330 and pH of 6.9). Green buffer was supplemented with 20% plasma egg yolk (osmolality of 335 and pH of 6.9). The osmolality and pH of INRA 96 were 310 and 7, respectively. Extenders at a ratio of 1:3 were added to semen followed by pipetting 10 times with an automatic pipettor. The water-jacketed extended specimen was covered with foam and transferred to individual vaccine carrier equipped with 4 ice packs. This system of cooling not only allows the specimen to cool down slowly and reach 4°C after 7 h, but also reduces the viscosity of camel semen. The assessment was carried out 7 and 24 h after semen dilution, when the specimen reached 4°C. Semen viability parameters were assessed after short-term semen preservation in different extenders. Total motility and progressive forward motility were examined subjectively by single operator using Sperm Track (ISAS, Proiser, Spain) after diluting the specimen to achieve 25 × 106 sperm mL−1. Live percentage of sperm was estimated using Eosin B Fast Green staining method. Plasma membrane integrity was assessed using the hypo-osmotic swelling (HOS) test. Following arcsin transformation, data were analysed by GLM procedure followed by Tukey test in SAS (SAS Institute Inc., Cary, NC, USA). At 7 and 24 h, there were no differences among the 3 extenders in total motility of sperm (Hashi: 73 and 67.4%; Green buffer: 71.6 and 62.1%; INRA 96: 70 and 66.2%; P > 0.05), live percentage of sperm (Hashi: 76 and 73%; Green buffer: 70.5 and 65.6%; INRA 96: 77.8 and 70.7%; P > 0.05), or HOS test (Hashi: 52.4 and 45.2%; Green buffer: 49.6 and 40.6%; INRA 96: 57.3 and 51.1%; P > 0.05). However, at the same times, progressive forward motility was similar between Hashi (47.7 and 28.6%) and Green buffer (40 and 23.5%; P > 0.05) but was different between Hashi and INRA 96 (23.6 and 16.7%; P < 0.05). In conclusion, Hashi and Green buffer could be considered suitable extenders to preserve Bactrian camel semen under chilled condition. Further studies with a larger number of bulls and ejaculates are warranted.

Production of invivo embryos for transfer in dromedary camel is a well-established practice, whereas freezing of these embryos is still an ongoing challenge. A common approach in evaluation of freeze–thawing method is achieved by studying invitro development of frozen–thawed embryos. However, not much is known about the development pattern of fresh dromedary embryos during incubation. The objectives of this study were to evaluate the usefulness of commercial holding media for easy short-term culture of these embryos and to provide preliminary insights on the growth and development of hatched blastocysts with different shapes. Recovered hatched blastocysts from superovulated donors were graded as transferable and non-transferable. Embryos with significant folding or crinkliness were further categorized as collapsed, whereas those with a round or oval appearance were categorized as spherical. Culture was performed in 500-μL drops at 38.5°C, 5% O2, 0–6% CO2, and maximum humidity in groups of 2 to 4. The 4 experimental media included culture medium (CM; TCM-199, 10% fetal calf serum (FCS), 0.3mM sodium pyruvate, 2.2mg mL−1 sodium bicarbonate), serum-supplemented holding medium (SSH; Syngro+10% FCS), serum-free holding medium (Syngro) and V-Onestep (Vitromed). In experiment 1, a total of 36 embryos were assigned to 4 groups and further development of the embryos was monitored up to 96h by morphological evaluations, identifying static and degenerating embryos on daily basis. In experiment 2, a total of 16 spherical and 16 collapsed embryos were cultured in SSH and CM and two-thirds of the culture drop was replaced with fresh medium at 72h. The proportion of developing embryos and their size expansion was compared between treatments by Fisher’s test and Mann–Whitney U test, respectively. Statistically similar proportions of embryos continued to develop in all media within the first 48h despite a numeric advantage in CM group; at 72h, the proportion of growing embryos was significantly higher in CM (77.8%) and SSH (66.6%) compared with SFH (11.1%) and OneStep (22.2%) (P&lt;0.05). None of the embryos in SFH and only 1 embryo in the V-Onestep group survived beyond 72h, whereas 3/9 embryos in SSH and 7/9 embryos in CM continued to expand. In experiment 2, the proportion of spherical embryos that developed was higher compared with their collapsed counterparts (8/8 in both groups vs. 5/8 and 4/8 in CM and SSH, respectively) at 24h. However, remaining collapsed embryos grew and expanded at similar rates to spherical ones in each group (P&gt;0.05). Replacing the medium did not favour continuation of embryonic growth in SSH beyond 72 h; only 5/16 embryos survived to 96h compared with 12/16 in CM. In conclusion, serum-supplemented commercial holding preparations provide comparable results to culture medium for short-term incubation of invivo dromedary embryos. Natural collapsing of hatched blastocysts might be associated with lower developmental competence.