Experiment 1: three variants of SLC

Frozen semen from four bulls in commercial semen production was kindly provided by Viking Genetics, Skara, Sweden. Four straws from each bull/batch (n = 16), were thawed at 37°C for 12 s and pooled. An aliquot of 200 μl was used as a control (unselected), and three variants of the SLC treatment were prepared following a protocol described previously (Morrell et al., Reference Morrell, Dalin and Rodriguez-Martinez2009; Anel-López et al., Reference Anel-López, Martínez-Rodríguez, Soler, Fernández-Santos, Garde and Morrell2015), with slight modifications. For each treatment an aliquot of 200 μl was layered on top of a column of Bovicoll (Androcoll-B) as follows: ‘Small’: 4 ml of colloid in a conical centrifuge tube; ‘Mini’: 1 ml of colloid in a conical centrifuge tube and ‘Mini-EP’: 1 ml of colloid in a 1.5 ml Eppendorf® tube. The tubes were centrifuged at 300 g for 20 min and each sperm pellet was transferred to a clean tube containing 20 μl of buffer B (patent applied for) with 5% BSA added to prevent the spermatozoa adhering to surfaces.

A Nucleocounter SP 100 (Chemometec, Allerød, Denmark) was used to evaluate the sperm concentration, according to Hansen et al. (Reference Hansen, Vermeiden, Vermeiden, Simmet, Day and Feitsma2006) with slight modifications (5 μl of the sample, instead of 50 μl and 500 μl of the detergent, instead of 5 ml).

Sperm motility was assessed in an aliquot of 5 μl on a pre-warmed microscope slide, by computer-assisted semen analysis (CASA) using the SpermVision™ (Minitűb, Tiefenbach, Germany) connected to an Olympus BX 51 microscope (Olympus, Japan). The yield of motile spermatozoa after SLC was calculated as follows (Morrell et al., Reference Morrell, Dalin and Rodriguez-Martinez2009):

$$\begin{eqnarray*} {\rm{Yield\ }}\left( {\rm{\% }} \right)\; =\;\frac{{{\rm{Total\ number\ of\ motile\ spermatozoa\ after\ SLC}}}}{{{\rm{Total\ number\ of\ motile\ spermatozoa\ before\ SLC}}}}{\rm{\ }} \times {\rm{\ }}100 \end{eqnarray*}$$}}

To obtain the percentage of sperm with functional membranes, the hypo-osmotic swelling test (HOST) was used, following the protocol described by Correa & Zavos (Reference Correa and Zavos1994). Ten microlitres of each sample were incubated in 100 μl of fructose–sodium citrate (100 mOsm) at 37°C for 1 h. After incubation, a 5 μl drop was placed on a slide with a cover glass and a total of 200 spermatozoa was evaluated by phase-contrast microscopy (Olympus BH2, Japan; ×400 magnification).

Chromatin integrity was evaluated using the sperm chromatin structure assay (SCSA). Aliquots of selected and unselected samples were mixed 1:1 with TNE buffer (Evenson & Jost, Reference Evenson and Jost2000), snap-frozen in liquid nitrogen and transferred to a −80°C freezer until analysed. The SCSA has been described in detail by Goodla et al. (Reference Goodla, Morrell, Yusnizar, Stålhammar and Johannisson2014). The DNA fragmentation index (%DFI) was calculated as the proportion of sperm fluorescing red out of the total population (sperm with red and sperm with green fluorescence) (Evenson & Jost, Reference Evenson and Jost2000).

Yield of motile spermatozoa, percentage of sperm with functional membranes (HOST) and %DFI, in the unselected and selected sperm samples were compared using repeated measures analysis of variance (ANOVA) (repeated over bulls) using R v3.2 (R Development Core Team, 2014).

Experiment 2: IVF trial

The aim of this experiment was to determine if SLC-selected spermatozoa were capable of fertilization. Straws of semen of a bull (with proven fertility in IVF) were thawed and treated using two methods: Mini-SLC and swim-up (control). Mini-SLC was chosen as sperm preparation technique based on the results from Experiment 1.

In vitro maturation and fertilization

The methods used have been described previously by Abraham et al. (Reference Abraham, Gustafsson, Ruete and Brandt2012) with slight modifications: 50 μg/ml of gentamycin replaced penicillin and streptomycin in the culture medium, and 0.5 μg/ml FSH and 0.1 μg/ml LH (Stimufol, PARTNAR Animal Health, Port Huron, Canada) was added to the maturation medium instead of 10 μg/ml of each. Bovine ovaries were collected at a slaughterhouse (Linköping) and transported at 33–35°C to the laboratory within 3 h. Cumulus–oocyte complexes (COCs) were aspirated from follicles between 3 to 8 mm in diameter and collected in search medium (HEPES-buffered medium 199 with 0.2% w/v BSA fraction V, 50 IU penicillin and 50 μg/ml streptomycin). Only good quality COCs were selected for maturation (according to Goodhand et al., Reference Goodhand, Staines, Hutchinson and Broadbent2000). Groups of 40 COCs were incubated in maturation media (TCM-199 with l-glutamine, 50 μg/ml of gentamycin, 0.5 μg/ml FSH and 0.1 μg/ml LH (Stimufol, PARTNAR Animal Health, Port Huron, Canada), and 0.4% w/v fraction V BSA for 24 h in a 5% CO₂ in air incubator, at 38.5°C. After maturation, COCs were washed with wash medium (modified Tyrode's albumin lactate pyruvate; mTALP, containing 0.3% w/v fraction V BSA) and pipetted, leaving 3–5 layers of cumulus cells around each oocyte; they were then transferred to wells with fertilization media (mTALP containing 0.6% w/v fatty acid-free BSA, 3 μg/ml heparin, 3 μg/ml penicillinamine 3 μg/ml epinephrine and 1.1 μg/ hypotaurine).

Semen from one bull, known to work well in IVF in our laboratory, was used. The spermatozoa were thawed and two different sperm selection techniques were performed: Mini-SLC and swim-up (as control). The Mini-SLC is described in Experiment 1, with the exception that fertilization medium was used instead of buffer B to resuspend the pellet obtained after centrifugation. The swim-up was performed in capacitation media for 45 min at 38.5°C in a 5% CO₂ in air incubator, as follows: semen was pipetted into a centrifuge tube and 1 ml of capacitation medium was carefully layered on top. The tube was placed in the incubator at an angle of 45° for 45 min to allow motile spermatozoa to swim-up into the capacitation medium.

After swim-up, the spermatozoa were washed and concentrated by centrifugation (300 g) for 7 min before resuspending the sperm pellet in fertilization medium. Motility was assessed after thawing and after selection and sperm concentration was measured in both groups. Spermatozoa were added to the oocytes at a concentration of 1 × 10⁶ spermatozoa/ml and incubated for 22 h (5% CO₂ at 38.5°C). Fertilized oocytes were then denuded from cumulus cells and spermatozoa by pipetting and cultured in synthetic oviduct fluid, (SOF; Takahashi & First, Reference Takahashi and First1992) in a humidified atmosphere of 5% CO₂, 5% O₂ and 90% N₂ at 38.5°C. At 44 h post fertilization, cleavage was checked and the number of embryos beyond the 2-cell stage was noted. The number of blastocysts developed by day 7 and day 8 was recorded. On day 8, all blastocysts were graded (according to Lindner & Wright, Reference Lindner and Wright1983), fixed with 4% paraformaldehyde overnight at 4°C and washed in PBS (with 1% PVA). Afterward, they were stained for 20 min with Hoechst 33342 (2.5 μg/ml), washed and mounted on glass slides with Vectashield. The number of nuclei was recorded for each blastocyst by two observers independently, using an epifluorescence microscopy (SM 510, Carl Zeiss AB, Jena, Germany).

The cleavage rates and blastocyst rates were calculated from the number of fertilized oocytes.

Cleavage rate, blastocyst rate and the total number of nuclei per blastocyst were compared using a paired t-test (R Development Core Team, 2014). Four batches of oocytes were prepared.