Andrea Galli’s scientific contributions

Animal welfare, particularly in pig, is a constraint for animal breeding. In this regard the Italian D.Lgs 53/2004 established that from 2013 sows and gilts shall be kept in groups during the four weeks after the service to one week before the expected farrowing time. Although its relevance is conceptually and extensively recognised, animal welfare is not yet fully characterized at an objective level and a protocol based on parameters for objectively evaluating animal welfare is still not available. The aim of this study was to evaluate a series of approaches in order to define informative markers associated with animal welfare, which allow an objective assessment of this status in the farm. Eight hundred pregnant sows reared in single and group-housing systems in a local farm were compared at different levels of investigation before and after the changeover imposed by law. Ethological, clinical, reproductive, metabolic and immunological measures were detected for each of the eight weeks of gestation (4 and 8-14). At the same time transcriptomic analysis of blood cells was conducted by OpenArray System on 224 genes involved in immunological response. The informativeness of the variables was studied by factor analysis and the effect of the housing system and the gestational week were studied by GLM. The odds ratio was calculated with the random component. Results showed that the group-housing system, contrary to the single-housing one, presented no stereotypy but significant increase in the frequency of lameness (P<0.001) and a decrease in fertility parameters (non-return to estrus 56-day post AI: 83% vs. 92%; farrowing rate: 78% vs. 88%, respectively) (P<0.05). The group-housing system revealed differences in the hematological picture for some parameters indicator of the metabolic status (ameliorative values for albumin, OR=4.4 and ALP OR=1.5 and pejorative for the AST, OR=0.6 and bilirubin, OR=0.4) when compared to the single, while at immunological level showed ameliorative values for bactericidal (OR=3.2) and complement (OR=24.3) and pejorative for lysozyme (OR=0.3) and C-reactive protein (P<0.001). At the functional genomics level, out of the 224 genes analyzed, 88 were under-expressed (Log FoldChange ≤1.5; P<0.05) in group-housing with respect to the single-housing system, within all the weeks and for all the gestational groups. Overall, the results of this multidisciplinary study provide potential candidates markers of sows housing relating stress.

The in vitro assessments of sperm quality represent an important, but limited, approach to evaluate ejaculated fecundant capacity. To date, the most popular method worldwide for fertility estimation is related to non-return (NR) rate which makes it possible to estimate fertility in vivo only after the widespread use of a bull. This work is aimed at evaluating possible existing differences in trascriptomic profile of spermatozoa of equal motility in bulls of contrasting proved fertility in order to produce more insights into the very origin of hypofertility. Ten Holstein Friesian bulls were selected on the basis of their 56-day NR to estrus and assigned to two groups of low (ERCR < -1.5) and high (ERCR > +1.5) estimated relative conception rates (90% reliability). Motile fraction of the frozen/thawed sperm was selected by discontinuous Percoll gradients (t0) and an aliquot was incubated at 37°C for one hour and then subjected to Percoll (t1). The spermatozoal RNA was extracted and purified from t0 and t1 motile sperm, reverse transcribed to ds-cDNA and subjected at Illumina sequencing. Unigene expression was calculated using the reads per kb per million reads method (RPKM). The total spermatozoal transcripts (FPKM>0) were classified into biological processes, cellular components and molecular functions, and 6 genes were found to be differentially expressed (P<0.05) between high and low fertility bulls both at t0 and t1. At to, actin beta (ACTB), the mitochondrially encoded NADH dehydrogenase subunit 1 (ND1) and subunit 2 (ND2), phospholipase C epsilon 1 (PCLE1) and the tumor protein translationally-controlled (TPT1) were up-regulated while the mitochondrial voltagedependent anion channel (VDAC2) was down-regulated in the high with respect to the low fertility groups. At t1 out of the same 6 genes PCLE1 was found to be still up-regulated whereas ACTB, ND1, ND2, VDAC2 and TPT1 were oppositely down-regulated in the high with respect to low fertility groups. These data provide a specific pattern of expression of these genes in motile spermatozoa of high and low fertility bulls at thawing and after incubation which conceptually reproduces the latent period of sperm in the female genital tracts after insemination. The analysis of gene expression would represent a new instrument of investigation that could potentially identify molecular markers for fertility to integrate conventional methodologies currently used to assess sperm fertilizing potential.

Laboratory assessments of sperm traits are poor indicators of fertility. Because the variability in the quality of oocytes collected from different donors drastically influence in vitro embryo production, the aim of this work was to implement an in vitro model to compare the developmental potential of embryos produced by bulls of contrasting in vivo fertility and ovocytes retrieved from the same donor in order to minimize the female related variability. For each trial (n=54), one pair of good quality ovaries of a same donor were split longitudinally and oocytes were recovered by slicing method. Thereafter, matured oocytes were fertilized with sperm of two bulls of low estimated relative conception rates (ERCR <-2) and high (ERCR >+2) contrasting field fertility (6 vs. 6 bulls). Cleavage and blastocyst formation rates were compared. Sperm of high fertility bulls (ERCR+) gave also higher fertilization rates (cleavage) in vitro when compared to low fertility (ERCR-) bulls (odd ratio=1.23). Conversely, the embryonic development to the blastocyst stage was reduced (odd ratio=0.84) in the ERCR+ with respect to the ERCR-bulls. This paradoxical result demonstrates that in vivo bull fertility hardly correlates with in vitro blastocyst yield, but confirms that early events occurring at fertilization are better indicators of the fertility potential. Furthermore, this experimental approach indicates that differences in embryo production rates between bulls of contrasting field fertility may not be outlined in vitro even when bulls are compared using oocytes with variability limited to the same donor.